Methods of enhancing immune response

ABSTRACT

The present invention relates to a method of enhancing immune response in a subject.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/425,766, filed Nov. 23, 2016; U.S. ProvisionalPatent Application No. 62/476,160, filed Mar. 24, 2017; and U.S.Provisional Patent Application No. 62/576,511, filed Oct. 24, 2017, thecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof, for simultaneous, separateor sequential use for promotion and/or enhancement of an immune responsein a subject; a pharmaceutical composition comprising such combination;a method of promoting and/or enhancing an immune response in a subjectcomprising administration of said RAD001 or BEZ235 or combination to asubject in need thereof; use of same for preparation of a medicament forthe promotion and/or enhancement of an immune response in a subject; anda commercial package thereto.

BACKGROUND OF THE INVENTION

mTOR is an evolutionarily conserved serine/threonine kinase that plays acentral role in integrating environmental cues in the form of growthfactors, amino acids, and energy. In the study of the immune system,mTOR is emerging as a critical regulator of immune function because ofits role in sensing and integrating cues from the immunemicroenvironment. With the greater appreciation of cellular metabolismas an important regulator of immune cell function, mTOR is proving to bea vital link between immune function and metabolism. mTOR has theability to direct the adaptive immune response, e.g. promotingdifferentiation, activation, and function in T cells, B cells, andantigen-presenting cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method comprising thestep of administering (a) a low dose of RAD001 or a pharmaceuticallyacceptable salt thereof; (b) a low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof; or (c) a combination thereof, to a patient,wherein the patient experiences fewer illnesses due to infection than apatient not administered RAD001 or a pharmaceutically acceptable saltthereof, BEZ235 or a pharmaceutically acceptable salt thereof, or acombination thereof.

In another aspect, the present invention relates to (a) RAD001, or apharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor simultaneous, separate or sequential use for the promotion and/orenhancement of an immune response in a subject.

In one aspect, the invention provides a pharmaceutical compositioncomprising (a) RAD001, or a pharmaceutically acceptable salt thereof;(b) BEZ235, or a pharmaceutically acceptable salt thereof; or (c) acombination thereof. In some embodiments, BEZ235, or a pharmaceuticallyacceptable salt thereof, is effective at promoting and/or enhancing animmune response in a subject, such as innate immunity. In someembodiments, the combination is jointly therapeutically effective atpromoting and/or enhancing an immune response in a subject.

In one aspect, the present invention provides a method of promotingand/or enhancing an immune response in subject comprising administeringto subject in need thereof (a) RAD001, or a pharmaceutically acceptablesalt thereof; (b) BEZ235, or a pharmaceutically acceptable salt thereof;or (c) a combination thereof in an amount effective at promoting and/orenhancing said immune response.

In one aspect, the present invention also provides a method of treatingan age-related condition, comprising administering to a subject in needthereof an effective amount of (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof to treat said age-relatedcondition.

In one aspect, the present invention provides the use of (a) RAD001, ora pharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the promotion and/or enhancement of an immune response in a subject,and for the preparation of a medicament for the promotion and/orenhancement of an immune response.

In one aspect, the present invention provides the use of (a) RAD001, ora pharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the treatment of an age-related condition in a subject.

In one aspect, the present invention provides a commercial packagecomprising as active ingredients (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof, together with instructionsfor the simultaneous, separate or sequential use thereof in thepromotion and/or enhancement of an immune response in a subject.

In one aspect, the present invention provides a commercial packagecomprising (a) RAD001, or a pharmaceutically acceptable salt thereof andinstructions for the simultaneous, separate or sequential use with (b)BEZ235, or a pharmaceutically acceptable salt thereof, in the promotionand/or enhancement of an immune response in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the increase in antibody titers to influenza vaccinestrains in RAD001 and/or BEZ235-treated versus placebo cohorts. Shown isthe increase in the ratio (4 weeks after vaccination/baseline) ingeometric mean titers (GMT) to each of the 3 influenza vaccine strains(A/H1N1 [A/California/7/2009], A/H3N2 [A/Texas/50/2012], or B[B/Massachusetts/2/2012]) in RAD001 and/or BEZ235-treated cohortsrelative to the placebo cohort. The black bold line indicates the 1.2fold increase in GMT ratios relative to placebo that is required in 2out of 3 influenza vaccine strains in order to meet the primary endpointof the study. Asterisks indicate that the probability that the increasein GMT relative to placebo exceeds 1.0 is 100%.

FIG. 2 shows BEZ235 alone or in combination with RAD001 decreases theincidence and severity of respiratory tract infection during the 6 weekssubjects were treated with study drug. BEZ235 alone or in combinationwith RAD001 decreases the incidence and severity of respiratory tractinfection during the 6 weeks subjects were treated with study drug.(Top) The percentage of subjects in each cohort who reported having oneor more respiratory tract infections during the 6 weeks they weretreated with study drug is shown. (Bottom) The percentage of subjectswho experienced respiratory tract infections of moderate severity duringthe 6 weeks they were treated with study drug is shown. No subjects inthe study experienced respiratory tract infections that were assessed assevere. The numbers on top of each bar indicate the percent changerelative to placebo.

FIG. 3 shows that BEZ235 and RAD001+BEZ235 but not placebo treatmentleads to upregulation of immune system and interferon-induced geneexpression. Pathway enrichment analysis of gene expression changes inwhole blood before versus after 6 weeks of study drug treatment isshown. This analysis revealed a highly significant enrichment ofpathways related to immune system and interferon signaling in the BEZ235monotherapy and BEZ235+RAD001 combination cohort but not in the placebocohort. The X axis indicates the mean log 2 fold change in expression ofgenes in each pathway. The y axis indicates the −log 10 of the p valueof pathway upregulation, with a value greater than 6 indicatingstatistical significance with a Bonferroni false discovery rate of lessthan 0.05. Each dot represents a specific biological pathway.

FIG. 4 shows that BEZ235 has unacceptably high PK variability at dosesabove 10 mg. Dose (mg) vs AUClast are shown. Each dot represents theBEZ235 AUC last on either day 1 or day 8 of dosing for each individualpatient in each cohort.

FIG. 5 shows that BEZ235 alone or in combination with RAD001 upregulateextracellular matrix remodeling proteins.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

In one aspect, the present invention relates to (a) RAD001, or apharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereof.In another aspect, the present invention provides a method of promotingand/or enhancing an immune response in a subject, comprisingadministering to the subject an effective amount of (a) RAD001, or apharmaceutically acceptable salt thereof (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereofby simultaneous, separate or sequential administration.

The general terms used herein are defined with the following meanings,unless explicitly stated otherwise:

The terms “comprising” and “including” are used herein in theiropen-ended and non-limiting sense unless otherwise noted.

The terms “a” and “an” and “the” and similar references in the contextof describing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

The term “combination” or “pharmaceutical combination” is defined hereinto refer to either a fixed combination in one dosage unit form, anon-fixed combination or a kit of parts for the combined administrationwhere RAD001, or pharmaceutically acceptable salt thereof, and BEZ235,or pharmaceutically acceptable salt thereof may be administeredindependently at the same time or separately within time intervals thatallow that the combination partners show a cooperative, e.g.,synergistic, effect.

The term “fixed combination” means that the active ingredients ortherapeutic agents, e.g. RAD001 and BEZ235, are administered to apatient simultaneously in the form of a single entity or dosage form.

The term “non-fixed combination” means that the active ingredients ortherapeutic agents, e.g. RAD001 and BEZ235, are both administered to apatient as separate entities or dosage forms either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the subject, e.g., a mammal or human, in needthereof.

The term “pharmaceutical composition” is defined herein to refer to amixture or solution containing at least one therapeutic agent to beadministered to a subject, e.g., a mammal or human, in order to treat aparticular disease or condition affecting the subject thereof.

The term “pharmaceutically acceptable” is defined herein to refer tothose compounds, biologic agents, materials, compositions and/or dosageforms, which are, within the scope of sound medical judgment, suitablefor contact with the tissues a subject, e.g., a mammal or human, withoutexcessive toxicity, irritation allergic response and other problemcomplications commensurate with a reasonable benefit/risk ratio.

The terms “combined administration” as used herein are defined toencompass the administration of the selected therapeutic agents to asingle subject, e.g., a mammal or human, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “treating” or “treatment” as used herein comprises a treatmentrelieving, reducing or alleviating at least one symptom in a subject oraffecting a delay of progression of a disease, condition and/ordisorder. For example, treatment can be the diminishment of one orseveral symptoms of a disorder or complete eradication of a disorder.Within the meaning of the present invention, the term “treat” alsodenotes to arrest, delay the onset (i.e., the period prior to clinicalmanifestation of a disease) and/or reduce the risk of developing orworsening a disease.

The term “jointly therapeutically active” or “joint therapeutic effect”as used herein means that the therapeutic agents may be given separately(in a chronologically staggered manner, for example in asequence-specific manner) such that the warm-blooded animal (forexample, human) to be treated, still shows a interaction, such as asynergistic interaction (joint therapeutic effect). Whether this is thecase can, inter alia, be determined by following the blood levels,showing that both therapeutic agents are present in the blood of thehuman to be treated at least during certain time intervals.

An “effective amount”, “pharmaceutically effective amount”, or“therapeutically effective amount” of a therapeutic agent is an amountsufficient to provide an observable improvement over the baselineclinically observable signs and symptoms of the promotion and/orenhancement of the immune response.

The term “synergistic effect” as used herein refers to action of twoagents such as, for example, (a) RAD001, or a pharmaceuticallyacceptable salt thereof, and (b) BEZ235, or a pharmaceuticallyacceptable salt thereof, producing an effect, for example, promotingand/or enhancing an immune response in a subject, which is greater thanthe simple addition of the effects of each drug administered bythemselves. A synergistic effect can be calculated, for example, usingsuitable methods such as the Sigmoid-Emax equation (Holford, N. H. G.and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), theequation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp.Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation(Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Eachequation referred to above can be applied to experimental data togenerate a corresponding graph to aid in assessing the effects of thedrug combination. The corresponding graphs associated with the equationsreferred to above are the concentration-effect curve, isobologram curveand combination index curve, respectively.

The term “subject” or “patient” as used herein includes animals, whichare capable of promoting and/or enhancing an immune response and/orhaving an age-related condition. Examples of subjects include mammals,e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice,rabbits, rats and transgenic non-human animals. In some embodiments, thesubject is a human, e.g., a human suffering from, at risk of sufferingfrom, or potentially capable of suffering from an age-related condition.

The term “about” or “approximately” shall have the meaning of within10%, for example within 5%, of a given value or range.

The term “promote” or “enhance” in the context of an immune responserefers to an increase in immune response, such as an increase in theability of immune cells to target and/or kill cancer cells, to targetand/or kill pathogens and pathogen infected cells, and protectiveimmunity following vaccination, among others. In some embodiments,protective immunity refers to the presence of sufficient immune response(such as antibody titers) to protect against subsequent infection by apathogen expressing the same antigen or protection against a newpathogen.

The terms “immunosenescence” or “immunosenescent” refer to a decrease inimmune function resulting in impaired immune response, e.g., to cancer,vaccination, infectious pathogens, among others. It involves both thehost's capacity to respond to infections and the development oflong-term immune memory, especially by vaccination. This immunedeficiency is ubiquitous and found in both long- and short-lived speciesas a function of their age relative to life expectancy rather thanchronological time. It is considered a major contributory factor to theincreased frequency of morbidity and mortality among the elderly.Immunosenescence is not a random deteriorative phenomenon, rather itappears to inversely repeat an evolutionary pattern and most of theparameters affected by immunosenescence appear to be under geneticcontrol. Immunosenescence can also be sometimes envisaged as the resultof the continuous challenge of the unavoidable exposure to a variety ofantigens such as viruses and bacteria.

Immunosenescence is a multifactorial condition leading to manypathologically significant health problems, e.g., in the agedpopulation. Age-dependent biological changes such as a decline infunction of hematopoietic stem cells, an increase in PD1+ lymphocytes, adecline in the function of phagocytes, macrophages, dendritic cells,monocytes, T cells, B cells and NK cells, and a decline in innate,cell-mediated or humoral immunity contribute to the onset ofimmunosenescence. In one aspect, immunosenescence can be measured in anindividual by measuring telomere length in immune cells (See, e.g., U.S.Pat. No. 5,741,677). Immunosenescence can also be determined bydocumenting in an individual a lower than normal number of naïve CD4and/or CD8 T cells, a decrease in early pro-B cells and pre-B cells, adecrease in T and B cell repertoire, an increase in the number ofPD1-expressing T cells, e.g., a lower than normal number of PD-1negative T cells, an increase in CD8+CD28neg T cells, an increase inCD57+ and/or KLRG1+ CD8+ T cells, an increase in the number ofLAG-3-positive T cells, a change in T cell surface glycoproteins, achange in antibody glycosylation, a change the glycosylation of proteinsexpressed intracellularly or on the surface of immune cells, an increasein ICOS, CTLA-4, Tim-3 and/or LAG-3 expressing CD4 T cells, or decreasedresponse to vaccination in a subjects they age.

The term “impaired immune response” refers to a state in which a subjectdoes not have an appropriate immune response, e.g., to cancer,vaccination, pathogen infection, among others. In some embodiments, asubject having an impaired immune response is predicted not to getprotective antibody titer levels following prophylactic vaccination, orin which a subject does not have a decrease in cell-mediated immunity ordisease burden after therapeutic vaccination. A subject can also have animpaired immune response if the subject has an impaired expression ofinnate immune response genes. A subject can also have an impaired immuneresponse if the subject is a member of a population known to havedecreased immune function or that has a history of decreased immunefunction such as the elderly, subjects undergoing chemotherapytreatment, asplenic subjects, immunocompromised subjects, or subjectshaving HIV/AIDS. Methods described herein allow for the treatment of animpaired immune response by administration of a low, immune enhancing,dose of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, such asRAD001.

The term “low, immune enhancing dose” when used in conjunction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing dose of mTOR inhibitor results ina decrease in the number or percentage of PD-1 positive T cells and/oran increase in the number or percentage of PD-1 negative T cells, or anincrease in the ratio of PD-1 negative T cells/PD-1 positive T cells. Inan embodiment, the low, immune enhancing dose of mTOR inhibitor resultsin an increase in the number of naive T cells. In an embodiment, thelow, immune enhancing dose of mTOR inhibitor results in one or more ofthe following:

an increase in the expression of interferon-induced genes in blood cells

a decrease in the percentage of T cells expressing the markers LAG-3,CTLA-4, ICOS or Tim-3;

an increase in the expression of one or more of the following markers:CD62Lhigh, CD127high, CD27+, and BCL2, e.g., on memory T cells, e.g.,memory T cell precursors;

a decrease in the expression of KLRG1 or CD57, e.g., on naïve or memoryT cells, e.g., memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62Lhigh, increased CD127high, increased CD27+, decreased KLRG1, andincreased BCL2;

wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15 but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, or at least 70 but no more than 90%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 80%, at least10 but no more than 80%, at least 15 but no more than 80%, at least 20but no more than 80%, at least 30 but no more than 80%, at least 40 butno more than 80%, at least 50 but no more than 80%, or at least 60 butno more than 80%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 70%, at least10 but no more than 70%, at least 15 but no more than 70%, at least 20but no more than 70%, at least 30 but no more than 70%, at least 40 butno more than 70%, or at least 50 but no more than 70%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 60%, at least10 but no more than 60%, at least 15 but no more than 60%, at least 20but no more than 60%, at least 30 but no more than 60%, or at least 40but no more than 60%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 50%, at least10 but no more than 50%, at least 15 but no more than 50%, at least 20but no more than 50%, at least 30 but no more than 50%, or at least 40but no more than 50%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 40%, at least10 but no more than 40%, at least 15 but no more than 40%, at least 20but no more than 40%, at least 30 but no more than 40%, or at least 35but no more than 40%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 30%, at least10 but no more than 30%, at least 15 but no more than 30%, at least 20but no more than 30%, or at least 25 but no more than 30%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at least 1, 2, 3, 4or 5 but no more than 35, at least 1, 2, 3, 4 or 5 but no more than 40%,or at least 1, 2, 3, 4 or 5 but no more than 45%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than90%.

As is discussed herein, the extent of mTOR inhibition can be expressedas the extent of P70 S6K inhibition, e.g., the extent of mTOR inhibitioncan be determined by the level of decrease in P70 S6K activity, e.g., bythe decrease in phosphorylation of a P70 S6K substrate. The level ofmTOR inhibition can be evaluated by a method described herein, e.g. bythe Boulay assay.

The term “promote” or “enhance” in the context of an immune responserefers to an increase in immune response, such as an increase in theability of immune cells to target and/or kill cancer cells, to targetand/or kill pathogens and pathogen infected cells, and protectiveimmunity following vaccination, among others. In some embodiments,protective immunity refers to the presence of sufficient immune response(such as antibody titers) to protect against subsequent infection by apathogen expressing the same antigen.

mTOR Inhibitors

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment an mTOR inhibitor is anallosteric inhibitor. In an embodiment an mTOR inhibitor is a catalyticinhibitor.

Allosteric mTOR inhibitors include the neutral tricyclic compoundrapamycin (sirolimus), rapamycin-related compounds, that is compoundshaving structural and functional similarity to rapamycin including,e.g., rapamycin derivatives, rapamycin analogs (also referred to asrapalogs) and other macrolide compounds that inhibit mTOR activity.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,O-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as, everolimus as described in U.S. Pat. No. 5,665,772 andWO94/09010 the contents of which are incorporated by reference. Othersuitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4′S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin, and40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in US RE44,768, e.g.temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, for example (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807, the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin, and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described in US2005/0101624, the contents of which are incorporated by reference.

In mammalian cells, the target of rapamycin (mTOR) kinase exists as amultiprotein complex described as the mTORC1 complex or mTORC2 complex,which senses the availability of nutrients and energy and integratesinputs from growth factors and stress signaling. The mTORC1 complex issensitive to allosteric mTOR inhibitors such as rapamycin, is composedof mTOR, GβL, and regulatory associated proteins of mTOR (raptor), andbinds to the peptidyl-prolyl isomerase FKBP12 protein (a FK506-bindingprotein 1A, 12 kDa). In contrast, the mTORC2 complex is composed ofmTOR, GβL, and rapamycin-insensitive companion proteins of mTOR(rictor), and does not bind to the FKBP12 protein in vitro.

The mTORC1 complex has been shown to be involved in proteintranslational control, operating as a growth factor and nutrientsensitive apparatus for growth and proliferation regulation. mTORC1regulates protein translation via two key downstream substrates: P70 S6kinase, which in turn phosphorylates ribosomal protein P70 S6, andeukaryotic translation initiation factor 4E binding protein 1 (4EBP1),which plays a key role in modulating eIF4E regulated cap-dependenttranslation. The mTORC1 complex regulates cell growth in response to theenergy and nutrient homeostasis of the cell, and the deregulation ofmTORC1 is common in a wide variety of human cancers. The function ofmTORC2 involves the regulation of cell survival via phosphorylation ofAkt and the modulation of actin cytoskeleton dynamics.

The mTORC1 complex is sensitive to allosteric mTOR inhibitors such asrapamycin and derivatives in large part due to rapamycin's mode ofaction, which involves the formation of an intracellular complex withthe FKBP12 and binding to the FKBP12-rapamycin binding (FRB) domain ofmTOR. This results in a conformational change in mTORC1 which isbelieved to alter and weaken the interaction with its scaffoldingprotein raptor, in turn impeding substrates such as P70 S6K1 fromaccessing mTOR and being phosphorylated. Rapamycin and rapalogues suchas RAD001 have gained clinical relevance by inhibiting hyperactivationof mTOR associated with both benign and malignant proliferationdisorders.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaoneand the following chemical structure

Everolimus is an FDA approved drug for the treatment of advanced kidneycancer and is being investigated in several other phase III clinicaltrials in oncology. Preclinical studies have shown that Everolimus isable to inhibit the proliferation of a wide variety of tumor cell linesboth in vitro and in vivo, presumably through the suppression ofrapamycin sensitive mTORC1 function. Everolimus, as a derivative ofrapamycin, is an allosteric mTOR inhibitor that is highly potent atinhibiting part of the mTORC1 function, namely P70 S6 kinase (P70 S6K)and the downstream P70 S6K substrate P70 S6. Allosteric mTOR inhibitorslike everolimus (and other rapamycin analogs) have little or no effectat inhibiting the mTORC2 pathway, or its resulting activation of Aktsignaling. Further examples of allosteric mTOR inhibitors includesirolimus (rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTOR inhibitors include zotarolimus(ABT578) and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more complete inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylati on and cap-dependent translation.

BEZ235 is a catalytic mTOR inhibitor, having the chemical name2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrileand the following chemical structure

BEZ235 may also be used in its monotosylate salt form. The synthesis ofBEZ235 is described in WO2006/122806, the contents of which are herebyincorporated by reference.

As a catalytic mTOR inhibitor, BEZ235 is capable of shutting down thecomplete function of mTORC1 complex, including both the rapamycinsensitive (phosphorylation of P70 S6K, and subsequently phosphorylationof P70 S6) and rapamycin insensitive (phosphorylation of 4EBP1)functions. BEZ235 has a differential effect according to the drugconcentration used, whereby mTORC1 inhibition predominates at a very lowconcentration (less than or equal to 10 nmol/L), mTORC1 and mTORC2inhibition predominates at low concentration (less than 200 nmol/L) butdual PI3K/mTOR inhibition at relatively higher concentrations(approximately 500 nmol/L).

The structure of the active ingredients identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g., PatentsInternational (e.g, IMS World Publications). The corresponding contentthereof is hereby incorporated by reference.

In some embodiments, the invention provides a combination comprising (a)RAD001 or a pharmaceutically acceptable salt thereof, and (b) BEZ235 ora pharmaceutically acceptable salt thereof, for simultaneous, separateor sequential use for the enhancement or promotion of an immune responsein a subject.

In some embodiments, RAD001 is in the neutral form. In some embodiments,BEZ235 is the monotosylate salt. In some embodiments, RAD001 isadministered in a dosage range from 0.01-0.2 mg, e.g. 0.1 mg. In someembodiments, BEZ235 is administered in a dosage range from 1-20 mg, e.g.10 mg.

In some embodiments, the compound(s) or combination thereof is in animmediate release dosage form. In some embodiments, the compound(s) orcombination thereof is administered once per week. In some embodiments,the compound(s) or combination thereof is administered once per day.

In some embodiments, the subject is immunocompromised. In someembodiments, the subject is HIV+ or has AIDS. In some embodiments, thesubject has an infectious disease.

In some embodiments, the subject has an impaired immune response. Insome embodiments, the subject is immunosenescent. In some embodiments,the subject has an age-related condition, e.g. immunosenescence.

In one embodiment of the invention, the invention provides apharmaceutical composition comprising (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof or (c) a combination thereof and at least onepharmaceutically acceptable carrier.

Methods of Treatment

In one aspect, the present invention provides a method comprising thestep of administering (a) a low dose of RAD001 or a pharmaceuticallyacceptable salt thereof; (b) a low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof; or (c) a combination thereof, to a patient,wherein the patient experiences fewer illnesses due to infection than apatient not administered RAD001 or a pharmaceutically acceptable saltthereof, BEZ235 or a pharmaceutically acceptable salt thereof, or acombination thereof.

In some embodiments, the patient's innate immunity is enhanced.

In some embodiments, antigen-specific immunity is not enhanced.

In some embodiments, at least one interferon-inducing gene (ISG) isupregulated.

In some embodiments, the infection is a urinary tract infection.

In some embodiments, the infection is an infection of the teeth or gums.

In some embodiments, the infection is a respiratory tract infection.

In some embodiments, the infection is a viral infection.

In some embodiments, the patient is elderly.

In some embodiments, the patient is at least 65 years old.

In some embodiments, the patient is at least 75 years old.

In some embodiments, the patient is at least 85 years old.

In some embodiments, the low dose of RAD001 or a pharmaceuticallyacceptable salt thereof, the low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof, or combination thereof, is administered to thepatient for up to about 6 consecutive weeks; up to about 8 consecutiveweeks; up to about 10 consecutive weeks; up to about 12 consecutiveweeks; up to about 16 consecutive weeks; up to about 20 consecutiveweeks; up to about 6 consecutive months; up to about 1 year; or as partof long-term treatment (indefinitely).

In some embodiments, the low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof is administered to the patient for up to about 3consecutive weeks; up to about 6 consecutive weeks; up to about 8consecutive weeks; up to about 10 consecutive weeks; up to about 12consecutive weeks; up to about 16 consecutive weeks; up to about 20consecutive weeks; up to about 24 consecutive weeks; or up to about 6consecutive months.

In some embodiments, the patient continues to experience fewer illnessesdue to infection from about 1 day until about 1 year after theadministration of the RAD001 or a pharmaceutically acceptable saltthereof, the BEZ235 or a pharmaceutically acceptable salt thereof, orcombination thereof.

In some embodiments, the patient continues to experience fewer illnessesdue to infection for at least 1 month and until about 1 year after theadministration of the RAD001 or a pharmaceutically acceptable saltthereof, the BEZ235 or a pharmaceutically acceptable salt thereof, orcombination thereof.

In some embodiments, the patient continues to experience fewer illnessesdue to infection for at least 3 months and until about 1 year after theadministration of the RAD001 or a pharmaceutically acceptable saltthereof, the BEZ235 or a pharmaceutically acceptable salt thereof, orcombination thereof.

In some embodiments, the patient continues to experience fewer illnessesdue to infection for at least 6 months after the administration of theRAD001 or a pharmaceutically acceptable salt thereof, the BEZ235 or apharmaceutically acceptable salt thereof, or combination thereof.

In some embodiments, the patient is administered the low dose of BEZ235or a pharmaceutically acceptable salt thereof as a monotherapy.

In some embodiments, the patient is administered the low dose of RAD001or a pharmaceutically acceptable salt thereof as a monotherapy.

In some embodiments, the patient is administered both the low dose ofRAD001 or a pharmaceutically acceptable salt thereof and the low dose ofBEZ235 or a pharmaceutically acceptable salt thereof as a combinationtherapy.

In some embodiments, the pharmacokinetic AUC variability of patientsreceiving the low dose of BEZ235 or a pharmaceutically acceptable saltthereof is lower than a patient receiving a higher dose of BEZ235 or apharmaceutically acceptable salt thereof.

In some embodiments, RAD001 or a pharmaceutically acceptable saltthereof is in the neutral form.

In some embodiments, BEZ235 or a pharmaceutically acceptable saltthereof is the monotosylate salt.

In some embodiments, the method comprises the administration of 0.01-0.2mg of RAD001 or a pharmaceutically acceptable salt thereof.

In some embodiments, the method comprises the administration of 1-50 mgof BEZ235 or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is immunocompromised.

In some embodiments, the subject has an impaired immune response.

In some embodiments, the subject is immunosenescent.

In one aspect, the present invention provides a method of upregulatingat least one interferon-inducing gene (ISG), comprising administering(a) a low dose of RAD001 or a pharmaceutically acceptable salt thereof;(b) a low dose of BEZ235 or a pharmaceutically acceptable salt thereof;or (c) a combination thereof, to a patient.

In one aspect, the present invention provides a method of upregulatingat least one protein involved in extracellular matrix remodeling,comprising administering (a) a low dose of RAD001 or a pharmaceuticallyacceptable salt thereof; (b) a low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof; or (c) a combination thereof, to a patient.

In some embodiments, the protein involved in extracellular matrixremodeling is significantly upregulated following treatment for at least1 day, about 1 week, about 3 weeks, about 4 weeks, about 5 weeks, about6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks,about 20 weeks, about 24 weeks, or about 6 months.

In some embodiments, the present invention provides a method of treatinga disease or condition associated with aberrant extracellular matrixremodeling, comprising administering (a) a low dose of RAD001 or apharmaceutically acceptable salt thereof; (b) a low dose of BEZ235 or apharmaceutically acceptable salt thereof; or (c) a combination thereof,to a patient.

In some embodiments, the disease or condition associated with aberrantextracellular matrix remodeling is selected from heart failure, such asheart failure with preserved ejection fraction, chronic renal failure,glomerunephropathy, skin aging, NASH, hepatitis fibrosis/cirrhosis,pulmonary fibrosis including idiopathic pulmonary fibrosis,aging-related tendon dysfunction/stiffening, arthritis includingosteoarthritis, sarcopenia, myelofibrosis, myelodysplasia, aging-relateddysfunction of the blood brain barrier, diabetic nephropathy,atherosclerosis, or wound healing.

In some embodiments, the invention provides a method of promoting orenhancing an immune response in a subject comprising administering tosaid subject (a) RAD001, or a pharmaceutically acceptable salt thereof;(b) BEZ235, or a pharmaceutically acceptable salt thereof; or (c) acombination thereof in an amount which is therapeutically effective orjointly therapeutically effective at promoting or enhancing an immuneresponse.

In some embodiments, the invention provides a method of treating anage-related condition in a subject comprising administering to saidsubject (a) RAD001, or a pharmaceutically acceptable salt thereof; (b)BEZ235, or a pharmaceutically acceptable salt thereof; or (c) acombination thereof in a quantity which is therapeutically effective orjointly therapeutically effective to treat, prevent, or ameliorate anage-related condition, e.g. immunosenescence.

In some embodiments, the invention provides the use of (a) RAD001, or apharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the preparation of a medicament for the promotion or enhancement ofan immune response.

In some embodiments, the invention provides the use of (a) RAD001, or apharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the preparation of a medicament for the treatment of an age-relatedcondition, e.g. immunosenescence.

In some embodiments, the invention provides a commercial packagecomprising RAD001, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withBEZ235, or a pharmaceutically acceptable salt thereof, in the promotionor enhancement of an immune response.

In some embodiments, the invention provides a commercial packagecomprising RAD001, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withBEZ235, or a pharmaceutically acceptable salt thereof, in the treatmentof an age-related condition.

In some embodiments, a benefit of the use of the combination is thatlower doses of (a) RAD001, or a pharmaceutically acceptable salt thereofand (b) BEZ235, or a pharmaceutically acceptable salt thereof, when usedin combination can be used, for example, that the dosages need not onlyoften be smaller, but are also applied less frequently, or exhibitdiminished incidence of side-effects observed with larger doses or withone of the combination partners alone. This is in accordance with thedesires and requirements of the patients to be treated.

It can be shown by established test models that administration of (a)RAD001, or a pharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereofresults in the beneficial effects described herein before. The personskilled in the art is fully enabled to select a relevant test model toprove such beneficial effects. The pharmacological activity of acompound or combination may, for example, be demonstrated in a clinicalstudy or in an in vivo or in vitro test procedure as known in the art oras described hereinafter.

In one aspect, the invention provides a pharmaceutical compositioncomprising a quantity, which is jointly therapeutically effective atpromoting and/or enhancing an immune response in a subject, of (a)RAD001, or a pharmaceutically acceptable salt thereof; and (b) BEZ235,or a pharmaceutically acceptable salt thereof. In this composition, thecombination partners (a) and (b) are administered in a singleformulation or unit dosage form by any suitable route. The unit dosageform may also be a fixed combination.

In a further aspect, the invention provides pharmaceutical compositionsseparately comprising a quantity, which is jointly therapeuticallyeffective at promoting and/or enhancing an immune response in a subject,of (a) RAD001, or a pharmaceutically acceptable salt thereof and (b)BEZ235, or a pharmaceutically acceptable salt thereof which areadministered concurrently but separately, or administered sequentially.

The pharmaceutical compositions for separate administration of thecombination partners, or for the administration in a fixed combination,i.e. a single galenical composition comprising (a) RAD001, or apharmaceutically acceptable salt thereof; and (b) BEZ235, or apharmaceutically acceptable salt thereof, may be prepared in a mannerknown in the art and are those suitable for enteral (such as oral orrectal) and/or parenteral administration to subjects and comprising atherapeutically effective amount of at least one combination partneralone, e.g. as indicated above, or in combination with one or morepharmaceutically acceptable carriers.

The novel pharmaceutical composition may contain from about 0.1% toabout 99.9%, for example from about 1% to about 60%, of the activeingredient(s).

In some embodiments according to any of the preceding embodiments, thesubject is at least 65 years of age.

In some embodiments, the subject has congestive heart failure,

In some embodiments, the subject has diabetes mellitus.

In some embodiments, the subject has chronic renal failure.

In some embodiments, the subject is a current smoker.

In some embodiments, the subject has COPD.

Chronic obstructive pulmonary disease (COPD) is a lung diseasecharacterized by chronic obstruction of lung airflow that interfereswith normal breathing and is not fully reversible. The more familiarterms “chronic bronchitis” and “emphysema” are no longer used, but arenow included within the COPD diagnosis. COPD is not simply a “smoker'scough” but an under-diagnosed, life-threatening lung disease. A COPDdiagnosis is confirmed by a simple test called spirometry, whichmeasures how deeply a person can breathe and how fast air can move intoand out of the lungs. Such a diagnosis should be considered in anypatient who has symptoms of cough, sputum production, or dyspnea(difficult or labored breathing), and/or a history of exposure to riskfactors for the disease. Where spirometry is unavailable, the diagnosisof COPD should be made using all available tools. Clinical symptoms andsigns, such as abnormal shortness of breath and increased forcedexpiratory time, can be used to help with the diagnosis. A low peak flowis consistent with COPD, but may not be specific to COPD because it canbe caused by other lung diseases and by poor performance during testing.Chronic cough and sputum production often precede the development ofairflow limitation by many years, although not all individuals withcough and sputum production go on to develop COPD.

In some embodiments, the subject resides in a nursing home facility.

In some embodiments, the subject is residing in an assisted livingfacility.

In some embodiments, the subject resides in a skilled nursing facility.

In some embodiments, the subject resides in a rehabilitation facility.

In some embodiments, the subject requires assistance with one or moreactivity of daily living.

Activities of daily living (ADL) are routine activities that people tenddo every day without needing assistance. There are six basic ADLs:eating, bathing, dressing, toileting, transferring (walking) andcontinence. An individual's ability to perform ADLs is important fordetermining what type of long-term care (e.g. nursing-home care or homecare) and coverage the individual needs (i.e. Medicare, Medicaid orlong-term care insurance).

ADLs (activities of daily living): the things we normally do in dailyliving including any daily activity we perform for self-care such ascooking, feeding, bathing, dressing, grooming, work, homemaking, andleisure. The ability or inability to perform ADLs can be used as a verypractical measure of ability/disability in many disorders.

In some embodiments, the subject has mobility disability.

Mobility disability or mobility impairment refers to the impairedability of a person to use one or more of his/her extremities, or adecrease in strength needed to walk, grasp, or lift objects. The use ofa wheelchair, crutches, or a walker may be utilized to aid in mobility.Mobility impairment may be caused by a number of factors, such asaging-related sarcopenia, disease, an accident, or a congenital disorderand may be the result from muscular, neuro-muscular or orthopaedicimpairments.

Pharmaceutical compositions comprising a disclosed compound orcombination, including fixed combinations or non-fixed combinations, forenteral or parenteral administration are, for example, those in unitdosage forms, such as sugar-coated tablets, tablets, capsules orsuppositories, or ampoules. If not indicated otherwise, these areprepared in a manner known in the art, for example by means of variousconventional mixing, comminution, granulating, sugar-coating,dissolving, lyophilizing processes, or fabrication techniques readilyapparent to those skilled in the art. It will be appreciated that theunit content of a combination partner contained in an individual dose ofeach dosage form need not in itself constitute an effective amount sincethe necessary effective amount may be reached by administration of aplurality of dosage units. It will be further appreciated that the unitcontent of a combination partner for parenteral administration maycontain a higher dosage amount of the combination partner which isdiluted to the effective dosage amount before administration.

A unit dosage form containing the combination of agents or individualagents of the combination of agents may be in the form of micro-tabletsenclosed inside a capsule, e.g. a gelatin capsule. For this, a gelatincapsule as is employed in pharmaceutical formulations can be used, suchas the hard gelatin capsule known as CAPSUGEL™, available from Pfizer.

The unit dosage forms of the present invention may optionally furthercomprise additional conventional carriers or excipients used forpharmaceuticals. Examples of such carriers include, but are not limitedto, disintegrants, binders, lubricants, glidants, stabilizers, andfillers, diluents, colorants, flavors, and preservatives. One ofordinary skill in the art may select one or more of the aforementionedcarriers with respect to the particular desired properties of the dosageform by routine experimentation and without any undue burden. The amountof each carrier used may vary within ranges conventional in the art. Thefollowing references which are all hereby incorporated by referencedisclose techniques and excipients used to formulate oral dosage forms.See The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al.,Eds., American Pharmaceuticals Association (2003); and Remington: theScience and Practice of Pharmacy, 20th edition, Gennaro, Ed., LippincottWilliams & Wilkins (2003).

These optional additional conventional carriers may be incorporated intothe oral dosage form either by incorporating the one or moreconventional carriers into the initial mixture before or during meltgranulation or by combining the one or more conventional carriers withthe granules in the oral dosage form. In the latter embodiment, thecombined mixture may be further blended, e.g., through a V-blender, andsubsequently compressed or molded into a tablet, for example amonolithic tablet, encapsulated by a capsule, or filled into a sachet.

Examples of pharmaceutically acceptable disintegrants include, but arenot limited to, starches; clays; celluloses; alginates; gums;cross-linked polymers, e.g., cross-linked polyvinyl pyrrolidone orcrospovidone, e.g., POLYPLASDONE XL™ from International SpecialtyProducts (Wayne, N.J.); cross-linked sodium carboxymethylcellulose orcroscarmellose sodium, e.g., AC-DI-SOL™ from FMC; and cross-linkedcalcium carboxymethylcellulose; soy polysaccharides; and guar gum. Thedisintegrant may be present in an amount from about 0% to about 10% byweight of the composition. In one embodiment, the disintegrant ispresent in an amount from about 0.1% to about 5% by weight ofcomposition.

Examples of pharmaceutically acceptable binders include, but are notlimited to, starches; celluloses and derivatives thereof, for example,microcrystalline cellulose, e.g., AVICEL PH™ from FMC (Philadelphia,Pa.), hydroxypropyl cellulose hydroxylethyl cellulose andhydroxylpropylmethyl cellulose METHOCEL™ from Dow Chemical Corp.(Midland, Mich.); sucrose; dextrose; corn syrup; polysaccharides; andgelatin. The binder may be present in an amount from about 0% to about50%, e.g., 2-20% by weight of the composition.

Examples of pharmaceutically acceptable lubricants and pharmaceuticallyacceptable glidants include, but are not limited to, colloidal silica,magnesium trisilicate, starches, talc, tribasic calcium phosphate,magnesium stearate, aluminum stearate, calcium stearate, magnesiumcarbonate, magnesium oxide, polyethylene glycol, powdered cellulose andmicrocrystalline cellulose. The lubricant may be present in an amountfrom about 0% to about 10% by weight of the composition. In oneembodiment, the lubricant may be present in an amount from about 0.1% toabout 1.5% by weight of composition. The glidant may be present in anamount from about 0.1% to about 10% by weight.

Examples of pharmaceutically acceptable fillers and pharmaceuticallyacceptable diluents include, but are not limited to, confectioner'ssugar, compressible sugar, dextrates, dextrin, dextrose, lactose,mannitol, microcrystalline cellulose, powdered cellulose, sorbitol,sucrose and talc. The filler and/or diluent, e.g., may be present in anamount from about 0% to about 80% by weight of the composition.

The optimum ratios, individual and combined dosages, and concentrationsof the therapeutic agent or agents that yield efficacy without toxicityare based on the kinetics of the therapeutic agent's availability totarget sites, and are determined using methods known to those of skillin the art.

In accordance with the present invention, a therapeutically effectiveamount of each of (a) RAD001, or a pharmaceutically acceptable saltthereof; (b) BEZ235, or a pharmaceutically acceptable salt thereof; or(c) a combination thereof may be administered simultaneously orsequentially and in any order, and the components may be administeredseparately or as a fixed combination. For example, in one aspect theinvention provides a method of preventing or treating an infection orage-related disease according to the invention may comprise (i)administration of the first agent (a) in free or pharmaceuticallyacceptable salt form, and (ii) administration of an agent (b) in free orpharmaceutically acceptable salt form, simultaneously or sequentially inany order, in jointly therapeutically effective amounts, in someembodiments in synergistically effective amounts, e.g. in daily orintermittent dosages corresponding to the amounts described herein. Theindividual therapeutic agents may be administered separately atdifferent times during the course of therapy or concurrently in dividedor single combination forms. Furthermore, the term “administering” alsoencompasses the use of a pro-drug of a therapeutic agent that convertsin vivo to the therapeutic agent. The instant invention is therefore tobe understood as embracing all such regimens of simultaneous oralternating treatment and the term “administering” is to be interpretedaccordingly.

The effective dosage of each of the therapeutic agents or combinationthereof may vary depending on the particular therapeutic agent orpharmaceutical composition employed, the mode of administration, thecondition being treated, and the severity of the condition beingtreated. Thus, the dosage regimen is selected in accordance with avariety of factors including the route of administration and the renaland hepatic function of the patient. A clinician or physician ofordinary skill can readily determine and prescribe the effective amountof the single active ingredients required to alleviate, counter orarrest the progress of the condition.

In embodiments where two therapeutic agents are used in combination, theeffective dosage of each of the therapeutic agents may require morefrequent administration of one of the therapeutic agent(s) as comparedto the other therapeutic agent(s) in the combination. Therefore, topermit appropriate dosing, packaged pharmaceutical products may containone or more dosage forms that contain the combination of compounds, andone or more dosage forms that contain one of the combination oftherapeutic agent(s), but not the other therapeutic agent(s) of thecombination.

When the combination of therapeutic agents, such as a combination of (a)RAD001, or a pharmaceutically acceptable salt thereof; and (b) BEZ235,or a pharmaceutically acceptable salt thereof are applied in the form asmarketed as single drugs, their dosage and mode of administration can bein accordance with the information provided on the package insert of therespective marketed drug, if not mentioned herein otherwise.

In some embodiments, RAD001, for example its free form, is administeredorally at a dose in the range from about 0.01 mg to about 1 mg dailyand/or weekly. In some embodiments, the dosage of RAD001, particularlyits free form, is administered orally at a dosage of 0.1 mg daily to anadult person.

In some embodiments, BEZ235 or a pharmaceutically acceptable saltthereof, for example its p-toluenesulfonate salt, is administered orallyat a dose in the range from about 1 mg to about 20 mg daily or about 1mg to about 50 mg daily and/or weekly. In some embodiments, the dosageof BEZ235 or a pharmaceutically acceptable salt thereof, such as itsp-toluenesulfonate salt, is administered, for example orally, at adosage of about 10 mg daily to an adult person, such as a person aged 65years or older. In some embodiments, the dosage of BEZ235 or apharmaceutically acceptable salt thereof is from about 2 mg to about 19mg, about 3 mg to about 17 mg, about 4 mg to about 16 mg, about 5 mg toabout 15 mg, about 6 mg to about 14 mg, about 7 mg to about 13 mg, about8 mg to about 12 mg, or about 9 mg to about 11 mg. In some embodiments,the dose of BEZ235 or a pharmaceutically acceptable salt thereof isabout 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg,about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg,about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about35 mg, about 40 mg, about 45 mg, or about 50 mg. In some embodiments,BEZ235 is administered orally as its p-toluenesulfonate salt. In someembodiments, BEZ235 or a pharmaceutically acceptable salt thereof isadministered twice per day, once per day, once every two days, onceevery three days, once every four days, once every five days, once everysix days, once a week, once every two weeks, once every three weeks,once every four weeks, once a month, once every two months, once everythree months, once every four months, once every six months, or once peryear.

The optimal dosage of each therapeutic agent for promotion and/orenhancement of an immune response in a subject and/or treating anage-related condition in a subject can be determined empirically foreach individual using known methods and will depend upon a variety offactors, including, though not limited to, the degree of advancement ofthe disease; the age, body weight, general health, gender and diet ofthe individual; the time and route of administration; and othermedications the individual is taking. Optimal dosages may be establishedusing routine testing and procedures that are well known in the art.

The amount of each therapeutic agent that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the individual treated and the particular mode of administration.In some embodiments the unit dosage forms containing the combination oftherapeutic agents as described herein will contain the amounts of eachagent of the combination that are typically administered when thetherapeutic agents are administered alone.

Frequency of dosage may vary depending on the therapeutic agent used andthe particular condition to be treated. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated,which will be familiar to those of ordinary skill in the art.

In one aspect, the present invention provides a method of promotingand/or enhancing an immune response in a subject comprisingadministering to subject in need thereof (a) RAD001, or apharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereofin an amount which is therapeutically effective or jointlytherapeutically effective at promoting and/or enhancing an immuneresponse in a subject.

In another aspect, the present invention provides a method of treatingan age-related condition in a subject, comprising administering to asubject in need thereof an amount of (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof in an amount which istherapeutically effective or jointly therapeutically effective to treatan age-related condition, such as immunosenescence.

In another aspect, the present invention provides a method of promotingand/or enhancing an immune response in a subject comprisingadministering to subject in need thereof BEZ235, or a pharmaceuticallyacceptable salt thereof in an amount which is therapeutically effectiveat promoting and/or enhancing an immune response in a subject, such asan innate immune response.

In another aspect, the present invention provides a method of treatingan age-related condition in a subject, comprising administering to asubject in need thereof an amount of BEZ235, or a pharmaceuticallyacceptable salt thereof in an amount which is therapeutically effectiveto treat an age-related condition, such as immunosenescence.

In another aspect, the present invention provides the use of (a) RAD001,or a pharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the enhancement and/or promotion of an immune response and/or forthe preparation of a medicament for the enhancement and/or promotion ofan immune response.

In another aspect, the present invention provides the use of (a) RAD001,or a pharmaceutically acceptable salt thereof; (b) BEZ235, or apharmaceutically acceptable salt thereof; or (c) a combination thereoffor the treatment of an age-related condition and/or for the preparationof a medicament for the treatment of an age-related condition, such asimmunosenescence.

In another aspect, the present invention provides the use of BEZ235, ora pharmaceutically acceptable salt thereof for the enhancement and/orpromotion of an immune response and/or for the preparation of amedicament for the enhancement and/or promotion of an immune response,such as an innate immune response.

In another aspect, the present invention provides the use of BEZ235, ora pharmaceutically acceptable salt thereof for the treatment of anage-related condition and/or for the preparation of a medicament for thetreatment of an age-related condition, such as immunosenescence.

In one aspect, the present invention provides a commercial packagecomprising as active ingredients (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof together with instructionsfor the simultaneous, separate or sequential use thereof in theenhancement and/or treatment of an immune response.

In another aspect, the present invention provides a commercial packagecomprising as active ingredients (a) RAD001, or a pharmaceuticallyacceptable salt thereof; (b) BEZ235, or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof together with instructionsfor the simultaneous, separate or sequential use thereof in thetreatment of an age-related condition, such as immunosenescence.

In one aspect, the present invention provides a commercial packagecomprising RAD001, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withBEZ235 or a pharmaceutically acceptable salt thereof, in the enhancementand/or promotion of an immune response.

In another aspect, the present invention provides a commercial packagecomprising RAD001, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withBEZ235 or a pharmaceutically acceptable salt thereof, in the treatmentof an age-related condition, such as immunosenescence.

In another aspect, the present invention provides a commercial packagecomprising BEZ235, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withRAD001 or a pharmaceutically acceptable salt thereof, in the enhancementand/or promotion of an immune response, such as an innate immuneresponse.

In another aspect, the present invention provides a commercial packagecomprising BEZ235, or a pharmaceutically acceptable salt thereof, andinstructions for the simultaneous, separate or sequential use withRAD001 or a pharmaceutically acceptable salt thereof, in the treatmentof an age-related condition, such as immunosenescence.

In another aspect, the present invention provides a commercial packagecomprising BEZ235, or a pharmaceutically acceptable salt thereof, andinstructions for use in the enhancement and/or promotion of an immuneresponse, such as an innate immune response.

In another aspect, the present invention provides a commercial packagecomprising BEZ235, or a pharmaceutically acceptable salt thereof, andinstructions for use in the treatment of an age-related condition, suchas immunosenescence.

Pathogenic Infections

In another aspect, the methods provided herein can be used to treat orprevent an infection by a pathogen in a subject. In some embodiments,the subject is immunodeficient. In some embodiments, the subject isimmunosenescent. In some embodiments, the pathogen is a viral pathogen,e.g., a viral pathogen e.g. HIV, meningitis causing viruses,encephalitis causing viruses, Hepatitis A, Hepatitis B, Hepatitis C,rabies virus, polio virus, influenza virus, parainfluenza virus,adenovirus, rhinovirus, measles virus, mumps virus, rubella, pertussis,papilloma virus, yellow fever virus, respiratory syncytial virus,parvovirus, Norwalk virus, chikungunya virus, hemorrhagic fever virusesincluding Ebola virus, dengue virus, Zika virus, and Herpes viruses,e.g., varicella, cytomegalovirus and Epstein-Barr virus. In someembodiments, the infection is a viral infection, such as a chronic viralinfection. In some embodiments, a chronic viral infection is selectedfrom Hepatitis A, Hepatitis B, Hepatitis C, Epstein-Barr Virus, HIV,Cytomegalovirus, Herpes Simplex Virus 1, Herpes Simplex Virus 2, HumanPapillomavirus, Adenovirus, and Kaposi's Sarcoma-Associated Herpesvirus.In some embodiments, a chronic viral infection comprises HIV.

For example, Lichterfeld and colleagues observed that HIV-specific CD8+T-cells showed reduced telomere length and an increase in telomerelength and telomerase activity upon inhibition of PD-1 (see e.g.,Lichterfeld, M et al. (2008) Blood 112(9):3679-3687). In anotherexample, PD-1 was significantly upregulated in hepatitis C(HVC)-specific CD8+ cytotoxic T lymphocytes (see e.g., Golden-Mason, L(2007) J. Virol. 81(17): 9249-9258).

In some embodiments, a viral infection comprises a viral respiratorytract infection. In some embodiments, the viral respiratory tractinfection is an upper viral respiratory tract infection. In someembodiments, the viral respiratory tract infection is a lower viralrespiratory tract infection. In some embodiments, the viral respiratorytract infection is caused by a rhinovirus, coronavirus, influenza virus,respiratory syncytial virus (RSV), adenovirus, metapneumovirus,enterovirus, bocavirus paramyxovirus, and/or parainfluenza virus. Insome embodiments, a viral respiratory tract infection is pneumonia. Insome embodiments, a viral respiratory tract infection includes a lungabscess. In some embodiments, a viral respiratory tract infectionincludes bronchitis.

In some embodiments, the pathogen is a bacterial pathogen, e.g., abacterial pathogen selected from Meningococcus, Haemophilus,Pneumococcus, Staphylococcus, Streptococcus, Neisseria, Moraxella,Escherichia coli, Klebsiella, Pseudomonas, Enterobacter, Proteus,Serratia, Legionella, Salmonella, Shigella, Acinetobacer, Listeria,Chlamydia, and Mycobacterium, among others.

In some embodiments, the pathogen is a parasitic pathogen, e.g.,Toxoplasma, Leishmania and malaria, T. cruzii, Helminth, e.g.,Schistosoma.

In some embodiments, the pathogen is a yeast or fungal pathogen, e.g.,Candida, Cryptococcus, Coccidioides, Blastomyces, aspergillus, ormucormycetes.

Senescence and Other Disorders

In another aspect, the methods provided herein can be used to treatsenescence in a subject. As used herein, the term “senescence” is meantto include all types of aging. In some embodiments, senescence comprisesimmunodeficiency, for example immunosenescence. Immunosenescenceincludes reduced immune response to infection with age and results fromthymic involution in T-cell lineages, resulting in decreased T cellproduction and export (see e.g., Shimatani, K et al. (2009) PNAS 106(37):15807-15812). In some embodiments, there is an increase inpopulation of a bona fide age-dependent CD4+ or CD8+ T cell populationdefined by a persistent expression of PD-1, which inhibits T cellresponses to antigens (see e.g., Shimatani, K et al. (2009) PNAS 106(37):15807-15812; Nunes, C et al. (2012) Clinical Cancer Research18(3):678-687). In some embodiments, senescence comprises cellularsenescence, in which a cell no longer divides. In some embodiments,age-related immunosenescence comprises decreased production of naivelymphocytes by hematopoietic stem cells (Chen, Science Signaling, ra75,2009). Cellular senescence is correlated with the progressive shorteningof telomeres that occurs with each cell division or the intracellularexpression of p16. In some embodiments senescence comprises anage-related decrease in the function of neutrophils, lymphocytes, NKcells, macrophages and/or dendritic cells (see e.g. Boraschi D et al.(2013) Sci Transl Med 5(185):ps8; Kumar R and Burns E A. (2008) ExpertRev. Vaccines 7(4): 467-479.

EXEMPLIFICATION

The following Examples illustrate the invention described above; theyare not, however, intended to limit the scope of the invention in anyway. The beneficial effects of the pharmaceutical compounds,combinations, and compositions of the present invention can also bedetermined by other test models known as such to the person skilled inthe pertinent art.

Example 1: Data Supporting a Claim that BEZ235 does not Enhance AntigenSpecific Immunity but Decreases Infection Rates by Enhancing InnateImmunity Via Upregulation of Interferon-Induced Genes (ISGs)

A total of 264 elderly volunteers ≥65 years of age, without unstablemedical conditions, were enrolled in a randomized double blindplacebo-controlled trial. Subjects were randomly assigned to receiveplacebo or one of four oral mTOR inhibitor dosing regimens: RAD001: 0.5mg daily, RAD001 0.1 mg daily, BEZ235 10 mg daily, or a combination of0.1 mg RAD001 and 10 mg BEZ235 daily. Subjects were treated for 6 weekswith study drug and, after a 2 week drug-free interval, were given a2014 seasonal influenza vaccine (Fluvax®, CSL Biotherapies).Hemagglutination inhibition (HI) antibody titers to the 3 strains ofinfluenza in the influenza vaccine were measured in serum collected atbaseline and four weeks after influenza vaccination. Serum HI titerswere measured at Quest Diagnostics. The subjects were then followed foranother 9 months off study drug. The primary endpoint of the study was a1.2 fold increase relative to placebo in the hemagglutination inhibition(HI) geometric mean titer (GMT) ratio (GMT 4 weeks post vaccination/GMTat baseline) to at least 2 out of 3 influenza vaccine strains. Thisendpoint was chosen because an approximately 1.2-fold increase in theinfluenza GMT ratio induced by the MF-59 vaccine adjuvant was associatedwith a decrease in influenza illness (1).

Only the combination of low dose RAD001 (0.1 mg daily) and BEZ235 (10 mgdaily) met the primary endpoint of the study and resulted in astatistically significant greater than 1.2-fold increase in theinfluenza GMT ratio in 3/3 influenza vaccine strains (FIG. 1). RAD001monotherapy (0.1 mg or 0.5 mg daily) resulted in a statisticallysignificant greater than 1.2-fold increase in influenza GMT ratio inonly 1/3 influenza vaccine strains. BEZ235 monotherapy did not result inan increase in influenza GMT ratios to any of the 3 influenza vaccinestrains. These results suggest that a combination of a low doseallosteric (RAD001) and catalytic (BEZ235) mTOR inhibitor resulted ingreater improvement in influenza vaccination response than RAD001 orBEZ235 monotherapy.

As an additional assessment of immune function, the overall infectionrate in each treatment group was assessed by having subjects record anyinfections they experienced during the year following the initiation ofstudy drug treatment in a diary that was reviewed by study investigatorsat each study visit. The number of infections per person per year wassignificantly decreased relative to placebo in the subjects in theRAD001 0.1 mg+BEZ235 10 mg combination and in the BEZ235 monotherapytreatment groups (Table 1). In addition, the rate of urinary tractinfections (Table 2) and tooth and gum infections (Table 3) were lowerin the BEZ235 monotherapy and RAD001+BEZ235 treatment groups than in theplacebo or RAD001 monotherapy treatment groups.

TABLE 1 BEZ235 alone or in combination with RAD001 was associated with asignificant reduction in infection rates for one year after study drugadministration Infection rate/person for one year after study drugadministration RAD001 RAD001 0.1 mg and RAD001 0.1 mg 0.5 mg BEZ235 10mg BEZ235 10 mg daily daily daily daily Placebo 2.0 2.1 1.8* 1.7** 2.5*p = 0.015; **p = 0.003

TABLE 2 BEZ235 alone or in combination with RAD001 was associated with areduction in the percentage of subjects with urinary tract infectionsfor one year after study drug administration Percent of subjects withurinary tract infections during one year after study drug administrationRAD001 RAD001 0.1 mg and RAD001 0.1 mg 0.5 mg BEZ235 10 mg BEZ235 10 mgdaily daily daily daily Placebo N = 51 N = 46 N = 46 N = 53 N = 48 10 114 0 12

TABLE 3 BEZ235 alone or in combination with RAD001 was associated with areduction in the rate of tooth or gum infections for one year afterstudy drug administration Number of tooth or gum infections per personfor one year after study drug administration RAD001 RAD001 0.1 mg andRAD001 0.1 mg 0.5 mg BEZ235 10 mg BEZ235 10 mg daily daily daily dailyPlacebo .02 .02 .02 .04 .13

The rate of infections during the 6 weeks subjects were treated withstudy drug was also examined. The main infections that occurred duringthe 6 weeks of study drug treatment were respiratory tract infections.Both BEZ235 and RAD001+BEZ235 led to a reduction in the incidence andseverity of respiratory tract infections during the 6 weeks subjectswere treated with study drug (FIG. 2).

To explore the possible mechanisms by which the combination of low doseBEZ235 monotherapy or RAD001+BEZ235 combination therapy reducedinfection rates in the elderly, RNAseq gene expression profiling wasperformed in whole blood obtained from subjects at baseline and after 6weeks of either placebo, BEZ235 or RAD001+BEZ235 treatment. RNAseqanalysis of whole blood revealed a highly significant enrichment ofpathways related to interferon signaling (FIG. 3). Some of the geneswhose expression was most highly upregulated in the enriched pathwaysincluded a subset of Type 1 interferon-induced genes (ISGs) that play acritical role in the immune response to viruses (Table 4) (2). Thesefindings suggest that upregulation of a subset of immune system andinterferon-induced genes by low dose BEZ235 and/or BEZ235+RAD001combination therapy enhance immune function and thereby reduce infectionrates in the elderly.

Gene Expression Profiling by RNASeq

Peripheral venous blood samples were collected into PAXgene Blood RNAtubes. Total RNA was purified from PAXgene collected blood and thequality and yield of the isolated RNA assessed using an Agilent 2100Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). RNA-Seqlibraries were prepared from the RNA using the Illumina TruSeq StrandedTotal RNA with the Ribo-Zero Globin Kit. This kit performed the removalof ribosomal RNA and globin mRNA in a single step. The obtainedlibraries were sequenced using the Illumina HiSeq2500 platform inpaired-end mode to a read length of 2×76 base-pairs (bp). Images fromthe instrument were processed using the manufacturer's software togenerate FASTQ sequence files.

Read quality was assessed by running FastQC (version 0.10) on the FASTQfiles. Sequencing reads showed excellent quality, with a mean Phredscore higher than 30 for all base positions. An average of 100 million76 bp paired-end reads per sample were mapped to the Homo sapiens genome(version GRCh38) and RefSeq UCSC was used for human gene and transcriptannotation. An in-house gene quantification pipeline was used todetermine the number of counts mapping to each gene (Schuierer, S. &Roma, G. The exon quantification pipeline (EQP): a comprehensiveapproach to the quantification of gene, exon and junction expressionfrom RNA-seq data. Nucleic acids research (2016)). On average, 88% ofthe total reads were mapped to the genome or the transcripts, and 30% ofthe aligned reads mapped to expressed sequences. Samples with less thaneight million expressed reads were removed from further analysis, aswell as three samples demonstrating a strand imbalance.

RNASeq Differential Gene Expression Analysis

Any gene with an average expression of less than one count per millionin any treatment/time condition was considered undetectable and removedfrom further analysis. Count data for remaining genes was thennormalized in R Statistical Computing Software (v3.1.3) using the voomnormalization procedure located in the limma package (Law, C W, Chen, Y,Shi, W, and Smyth, G K (2014). Voom: precision weights unlock linearmodel analysis tools for RNA-seq read counts. Genome Biology 15, R29.).

When evaluating the technical quality of the data, we performed aprinciple component analysis using the prcomp function in R. Thisanalysis indicated that the first principle component reflected multipleaspects of sample processing (RNA extraction batch, yield of mRNA, etc).Thus, we included the value of this principle component as an adjustmentfor technical variation in subsequent analysis.

For each gene, the following linear mixed model was fit to thevoom-normalized expression data, adjusting for relevant covariates (age,gender, and BMI) as well as PC1 using SAS v9.3 software.

proc mixed data=exprsData anovaf;

class SubjID Day Trt Gender;

model log 2exprs=Day Trt Day*Trt PC1 Age Gender Bmi/solution

DDFM=KenwardRoger;

repeated Day/type=un subject=Subj ID;

by gene;

where log 2exprs refers to the voom-normalized expression measurement;Day refers to the timepoint the sample was taken (pre- or post-dose);Trt refers to the treatment group of the subject; PC1 refers to thevalue of PC1 for that sample; Age refers to the age of the subject;Gender refers to the sex of the subject; Bmi refers to the body massindex of the subject; and SubjID refers to the ID of the subject.Differential expression fold changes and p-values were obtained usingthe SAS estimate statement. This analysis resulted in gene expressionresponse signatures for each of the treatment arms (BEZ, RAD+BEZ, andPlacebo). These gene expression signatures consisted of the post- vspre-treatment fold change and p-values for each treatment.RNASeq Gene Expression Pathway Enrichment Analysis

For this analysis, we looked for up- or down-regulated skew of genes indefined biological pathways within each treatment response signature.Canonical pathways (c2.cp.v5.0.entrez.gmt) were downloaded from thecanonical signatures database(http://software.broadinstitute.org/gsea/msigdb/). Signatures weredefined as described in the “RNASeq Differential Expression Analysis”section. For each pathway and treatment response signature, a weightedKS test was performed as previously described (Subramanian et al. Geneset enrichment analysis: A knowledge-based approach for interpretinggenome-wide expression profiles. Proc. Nat. Acad. Sci. vol 102. no 43.15545-15550 2005) and the resulting −log 10(p-values) are plotted as afunction of mean fold change of the genes in the pathway in FIG. 3.

RNASeq Gene Expression—Designation of Up-Regulated Genes

Genes were considered up-regulated if their fold change was determinedto be an outlier using the Tukey method of outlier detection.Specifically, an up-regulated gene met the following criteria: log 2(FCpost- vs. pre-treatment) greater than Q3+1.5*(Q3−Q1) where Q3 is theupper (i.e. third) quartile and Q1 the lower (i.e. first) quartile oflog 2(FC) for each treatment.

TABLE 4 Upregulated pathway genes in the BEZ235 monotherapy and BEZ235 +RAD001 combination therapy cohorts Mean FC Genes p-Value of in Up- Path-regulation Treatment Pathway way Skew Up-regulated genes BezREACTOME_INTERFERON_GAMMA_SIGNALING 0.04 1e−15.8 FCGR1A; FCGR1B;HLA-DQA2 Bez REACTOME_INTERFERON_ALPHA_BETA_SIGNALING 0.03 1.00E−11  IFITM3 Bez REACTOME_INTERFERON_SIGNALING 0.03 1e−26.6 IFITM3; FCGR1A;FCGR1B; HLA-DQA2 Bez REACTOME_IMMUNE_SYSTEM 0.01 1e−54.1 IFITM3; LILRB4;TRIM9; FCGR1A; FCGR1B; CD274; HLA-DQA2; RAP1GAP; TLR5; C1QA; C1QB; C3;C4BPA; FDCD1LG2; KLHL13; CD80; CDK1 BezREACTOME_CYTOKINE_SIGNALING_IN_IMMUNE_SYSTEM 0.02 1e−36.1 IFITM3;FCGR1A; FCGR1B; HLA-DQA2; CDK1 Comb REACTOME_INTERFERON_GAMMA_SIGNALING0.06 1e−16.4 GBP5; GBP6; FCGR1A; FCGR1B; GBP1; GBP2; HLA-DRB5; IRF7;OAS3; PML; STAT1; OASL; SOCS3 CombREACTOME_INTERFERON_ALPHA_BETA_SIGNALING 0.09 1e−21.8 IFITM3; IFI6;GBP2; IFI27; IFIT2; IFIT1; IFIT3; IRF7; MX1; OAS3; XAF1; STAT1; STAT2;OASL; SOCS3; ISG15 Comb REACTOME_INTERFERON_SIGNALING 0.04 1e−39.9IFITM3; GBP5; GBP6; FCGR1A; FCGR1B; DOX58; IFI6; GBP1; GBP2; HLA-DRB5;IFI27; IFIT2; IFIT1; IFIT3; IRF7; MX1; OAS3; HERC5; PML; XAF1; EIF2AK2;STAT1; STAT2; OASL; SOCS3; UBE2L6; ISG15 Comb REACTOME_IMMUNE_SYSTEM0.02 1e−77.5 IFITM3; TRIM9; GBP5; GBP6; FCGR1A; FCGR1B; RNF182; FOXO3;DDX58; KIF4A; IFI6; GBP1; GBP2; UBE2S; CD274; HLA-DRB5; IFI27; IFIT2;IFIT1; IFIT3; IL1RN; IRF7; MX1; OAS3; HERC5; PML; XAF1; PPP2R5B;EIF2AK2; RAP1GAP; S100B; STAT1; STAT2; UBE2B; RILP; OASL; SOCS3; UBE2L6;AIM2; KIF23; ISG15; CDK1; MRC2; CDC34 CombREACTOME_CYTOKINE_SIGNALING_IN_IMMUNE_SYSTEM 0.03 1e−48.5 IFITM3; GBP5;GBP6; FCGR1A; FCGR1B; DDX56; IFI6; GBP1; GBP2; HLA-DRB5; IFI27; IFIT2;IFIT1; IFIT3; IL1RN; IRF7; MX1; OAS3; HERC5; PML; XAF1; EIF2AK2; STAT1;STAT2; OASL; SOCS3; UBE2L6; ISG15; CDK1

The finding that BEZ235 monotherapy did not improve the response toinfluenza vaccination but did decrease infection rates was entirelyunexpected. Influenza vaccination response is a readout ofantigen-specific adaptive immunity. Thus, these findings suggest thatBEZ235, unlike RAD001, does not improve the immune response to specificantigens. Instead, BEZ235, as well as the combination of BEZ235+RAD001,may reduce infection rates by improving the non-antigen-specific innateimmune response to multiple pathogens via upregulation of ISGexpression.

Example 2: RAD001+BEZ235 May have Greater Efficacy than Either RAD001 orBEZ235 Monotherapy for Improving Vaccination Response in the Elderly (anAging-Related Endpoint) Because the Combination Results in MoreSelective and Complete TORC1 Inhibition than Either Monotherapy

Aging may be due to perturbations of a discreet set of cell signalingpathways including the mTOR pathway. mTOR signals via two complexes:TORC1 and TORC2. Many of the beneficial effects of mTOR inhibition onaging in preclinical species may be mediated by inhibition of TORC1(3,4). In contrast, TORC2 inhibition has been associated withhyperglycemia, hypercholesterolemia and with decreased lifespan in malemice (4,5). In addition, several long-lived animal models have increasedrather than decreased TORC2 activity (6). Therefore TORC1-specificinhibition may be optimal for the treatment of aging-related conditionsin humans such as declining immune function. Rapalogs such as RAD001 area class of allosteric mTOR inhibitors that consistently inhibit only theS6K node downstream of the TORC1 complex (7). Low concentrations ofBEZ235 such as were achieved in serum of elderly subjects dosed with 10mg BEZ235 daily (approximately 10-20 nM) also consistently inhibit onlythe S6K node downstream of TORC1 (8). At higher doses, BEZ235 completelyinhibits TORC1, but also inhibits TORC2 (8,9). However, low doses ofcatalytic inhibitors in combination with low doses of RAD001synergistically inhibit most if not all nodes downstream of TORC1without inhibiting TORC2 activity (8,10). Our clinical findings suggestthat more complete and selective TORC1 inhibition achieved with acombination of low dose RAD001 and BEZ235 is associated with betterenhancement of influenza vaccine response in elderly subjects than lowdose RAD001 or BEZ235 monotherapy. A combination of low dose RAD001 andBEZ235 also may have better efficacy for other aging-related conditionsthan RAD001 and/or BEZ235 monotherapy due to the more complete andselective TORC1 inhibition of the combination.

Example 3: BEZ235 or BEZ235+RAD001 Can be Used to Safely Enhance ImmuneFunction and Decrease Infection Rates in the Elderly Via Low LevelUpregulation of a Subset of Interferon-Induced Genes (ISGs)

The magnitude of ISG upregulation in whole blood after BEZ235monotherapy or RAD001+BEZ235 combination treatment was small (1-9%). Alow level increase in ISG expression may be sufficient to enhance immunefunction and decrease infection rates in the elderly while avoiding theundesirable adverse events that occur in patients treated withinterferon who have a much larger increase in ISG induction (11). Thus,it is believed that low level ISG induction avoids the safety andtolerability issues associated with high level ISG induction in patientstreated with interferon therapy.

Example 4: A 6 Week Course of BEZ235 or BEZ235+RAD001 Leads toPersistent Improvements in Immune Function After Drug Discontinuation

BEZ235 monotherapy and the combination of RAD001+BEZ235 led to asignificant reduction in overall infection rates for a year despite thefact that study drug was discontinued after 6 weeks of treatment. Theresults suggest that mTOR inhibitor therapy may lead to persistentimprovements in immune function for months after drug discontinuation.Short courses of the mTOR inhibitor rapamycin have also been shown toextend lifespan in mice, supporting the concept that the beneficialeffects of transient mTOR inhibition persisted for months after drugdiscontinuation (12,13).

Example 5: PK Variability of BEZ235 Can be Mitigated Via theAdministration of Low Doses

BEZ235 was developed as a dual PI3K-mTOR catalytic inhibitor foroncology indications. However, high doses of BEZ235 used for oncologyindications (up to the maximum tolerated dose of 1200 mg daily) wereassociated with unacceptably high inter-patient variability (AUCtau,sspercent coefficient of variation (% CV)=113%). The PK variability may bedue in part to the low solubility of BEZ235 above pH 3. However, theinter-patient variability of BEZ235 10 mg daily was acceptable(AUClast,ss % CV=39%) perhaps because low doses of BEZ235 are able to becompletely absorbed in the low pH of the stomach. Increasing PKvariability of BEZ235 at doses above 10 mg daily is shown in FIG. 4.

Example 6: BEZ235 Alone or in Combination with RAD001 May have BroadAnti-Aging Effects Via Upregulation of Extracellular Matrix RemodelingProteins

Extracellular matrix remodeling is a common mechanism underlying adiverse set of anti-aging interventions (14). These findings suggestthat lack of turnover of the extracellular matrix contributes to aging,and that agents that promote extracellular matrix remodeling may havebeneficial effects on aging-related conditions. Serum proteomics suggestthat BEZ235 monotherapy or BEZ235+RAD001 combination therapy promoteextracellular matrix remodeling. Serum samples were obtained from fromelderly subjects at baseline or after 6 weeks of treatment with placebo,BEZ235 10 mg daily, or the combination of BEZ235 10 mg daily+RAD001 0.1mg daily. Both 6 weeks of BEZ235 monotherapy or the combination ofBEZ235+RAD001 but not placebo led to a highly significant upregulation(neglog 10 p value of 14.34 for BEZ235 and 13.32 for BEZ235+RAD001combination) in the expression of proteins involved in extracellularmatrix disassembly/remodeling (FIG. 5). These findings suggest thatBEZ235 monotherapy or the combination of BEZ235+RAD001 may have broadanti-aging effects by stimulating the remodeling of the extracellularmatrix. BEZ235 monotherapy or the combination of BEZ235+RAD001 may beparticularly efficacious in the treatment of aging-related conditionsassociated with aging-associated abnormalities in the extracellularmatrix of tissues including heart failure, chronic renal failure andskin aging.

Example 7: BEZ235 Alone or in Combination with RAD001 May DecreaseRespiratory Tract Infections (RTIs) in Elderly Subjects

In the US, pneumonia and influenza are the 5th leading cause of death inpeople aged 85 and over (National Vital Statistics Report, Deaths, FinalData for 2014, 2016). Moreover, respiratory viruses (for which currentlythere are no effective treatments) cause the majority of communityacquired pneumonias in this population (Jain et al 2015). In addition,7% of people ≥85 years of age in the US go to the emergency room withRTIs each year (Goto et al 2016). Finally, hospitalizations for RTIsincrease the risk of subjects age ≥85 getting admitted to a nursinghome. In the US, only 10% of people age 85-95 reside in a nursing home,but 36% of subjects ≥85 years who are hospitalized get discharged to anursing home (The Older Population 2010 US Census Briefs, NationalCenter for Health Statistics Data Brief 2015).

Prevention of RTIs are also an unmet medical need for elderly subject≥65 years of age with underlying risk factors such as chronicobstructive pulmonary disease (COPD), asthma, chronic bronchitis, type 2diabetes mellitus (T2DM), congestive heart failure (CHF), and currentsmoking. For instance, RTIs are the most common cause of COPD and asthmaexacerbations (Nicholson et al BMJ, 2003; Sethi et al 2008). RTIs arealso the underlying cause of 16% of hospital admissions in patients withcongestive heart failure (Chin and Goldman 1997). Patients with T2DMalso are at increased risk of lower respiratory tract infections (Mulleret al 2005) and have an increased risk of hospitalization with pneumonia(Kornum et al 2008). Current smoking also increases the risk ofdeveloping community acquired pneumonia (Almirall et al 1999). Finally,elderly subjects who have previously had pneumonia are at increased riskfor recurrent pneumonia (Hedlund et al 1992).

Accordingly, the purpose of this study is to evaluate the efficacy,tolerability and safety of BEZ235 alone or in combination with RAD001 tosupport dose selection for further development to reduce the incidenceof RTIs in elderly subjects at increased risk of RTI-related morbidityor mortality.

We will determine if low dose mTOR inhibitors decrease the incidence andseverity of RTIs in high risk elderly populations defined as age ≥85years or age ≥65 years who are current smokers, have underlying COPD,asthma, chronic bronchitis, CHF, and/or T2DM, and/or who have beenadmitted to the hospital or gone to an emergency room with a RTI in thepast 12 months.

Objectives and Related Endpoints

TABLE 5 Objectives and related Endpoints Objectives Endpoints PrimaryObjective Endpoint for Primary Objective  To assess the dose-responserelationship  The percentage of subjects who develop  of 2 differentdoses of BEZ235 alone or in  one or more RTIs through Week 16 as combination with RAD001 as measured  assessed by pre-specifieddiagnostic  by the percent of elderly subjects  criteria  experiencingone or more RTIs as  compared to placebo during 16 weeks of  treatment.Secondary Objectives Endpoints for Secondary Objectives  To assess thesafety and tolerability of 2  All safety endpoints (including adverse different doses of BEZ235 alone or in  events and safety laboratories)up until  combination with RAD001 in elderly  and including 8 weeks postdose.  subjects  The percentage of subjects who develop  To evaluate theefficacy of BEZ235 alone  one or more RTIs through Week 24 as  or incombination with RAD001  assessed by prespecified clinical  compared toplacebo in reducing the  diagnostic criteria  incidence of RTIs for 24weeks  Incidence of laboratory-confirmed viral  To evaluate the efficacyof BEZ235 alone  RTIs through Week 16 as assessed by  or in combinationwith RAD001  respiratory virus PCR of nasopharyngeal  compared toplacebo at decreasing the  swabs  incidence of laboratory-confirmedviral  To determine whether covariates e.g.,  RTIs for 16 weeks.  bodysize, age, gender, race, organ  To evaluate the pharmacokinetics (PK) of function) affect systemic exposure to  2 different doses of BEZ235given alone  BEZ225 and whether these covariates  or in combination withRAD001  should be used to adjust dosing in future  studies. ExploratoryObjectives Endpoints for Exploratory Objectives  To explore the effectof BEZ235 alone or  Percentage of subjects in each subgroup  incombination with RAD001 compared  who develop one or more RTIs through to placebo on the incidence of RTIs in  Weeks 16 and 24 as assessed by each of the following subsets of elderly  prespecified clinicaldiagnostic criteria  subjects:  Total number of infections per person  ≥85 years of age,  through Week 16 or Week 24 as assessed   ≥65 yearof age with:  by prespecified clinical diagnostic    Asthma or  criteria   COPD or chronic bronchitis or  Total number of UTIs per personthrough    T2DM or  Week 16 or Week 24 as assessed by    CHF or prespecified clinical diagnostic criteria    Current smoker or Severity and duration of upper    Emergency room visit or  respiratorytract symptoms as assessed    hospitalization for RTI within past  bythe short Wisconsin Upper    12 months  Respiratory Symptom Survey(WURSS-  To explore the effect of BEZ235 alone or  21)  in combinationwith RAD001 compared  Change in hemagglutination inhibition  to placeboon total infection rates for 16  (HI) geometric mean titer (GMT) from or 24 weeks  baseline to 4 weeks post influenza  To explore the effectof BEZ235 alone or  vaccination  in combination with RAD001 compared Number of visits to a health care  to placebo on urinary tractinfection (UTI)  provider for symptoms related to  rates for 16 or 24weeks  infections; total number of CXRs done to  To explore the effectof BEZ235 alone or  evaluate infections, total number of  in combinationwith RAD001 compared  antibiotics prescribed for infections; total  toplacebo at decreasing the severity or  number of visits to the ED forinfections;  duration of upper respiratory tract  and total number ofhospitalizations for  infection symptoms in the subset of  infectionsthrough Week 16 or 24 of the  subjects who develop upper respiratory study as assessed by review of medical  tract infections  records  Toexplore the effect of BEZ235 alone or  Change from baseline to Week 16in  in combination with RAD001 on the  6MWT  response to influenzavaccination in the  Change from baseline to Week 16 in grip  subset ofsubjects who receive influenza  strength as assessed by a dynamometer vaccination as part of their routine health  Change from baseline toWeek 16 in  care during the study  cardiac function as assessed by  Toexplore the effect of BEZ235 alone or  echocardiogram.  in combinationwith RAD001 compared  Change from baseline to Week 16 in  to placebo onhealth care resource  perception of breathlessness as assessed utilization for infections  by a questionnaire administered before  Toexplore the effect of BEZ235 alone or  and after a 6 MWT  in combinationwith RAD001 compared  Change from baseline to Week 16 in  to placebo onimprovement in physical  gene expression in whole blood and in  functionas assessed by the 6 minute walk  soluble biomarkers in serum  test(6WMT)  To explore the effect of BEZ235 alone or  in combination withRAD001 compared  to placebo on grip strength  To explore the effect ofBEZ235 alone or  in combination with RAD001 compared  to placebo oncardiac function  To explore the effect of BEZ235 alone or  incombination with RAD001 as  compared to placebo on the perception of breathlessness  To explore the effect of BEZ235 alone or  incombination with RAD001 compared  to placebo on gene expression and circulating biomarkers in bloodStudy Design

This is a 24 week randomized, blinded, placebo-controlled,parallel-group, dose finding 2-stage adaptive design study to assess thesafety, tolerability and efficacy of two doses of BEZ23 5 alone or incombination with RAD001 as compared to placebo in elderly subjectswithout unstable medical conditions. Subjects can be included if theyare at increased risk of a respiratory tract infection as defined bybeing either ≥85 years of age or ≥65 years of age and a current smoker,or with underlying COPD, asthma, chronic bronchitis, T2DM, or CHF and/orhave been hospitalized or evaluated in an emergency room in the pastyear for a RTI. The study will be composed of a 6 week screening period,a 4 week run-in period, 16-week treatment period and an 8 week follow-upperiod

Study Periods: The total planned duration of the study for each subjectis up to 24 weeks from randomization to the last follow up visit (FIG.3-1); study treatment is taken for 16 weeks.

Screening Period: During the screening visit (maximum 6 weeks prior tobaseline/randomization), subjects will be assessed for eligibility toparticipate in the trial based on inclusion/exclusion criteria.

Run-in Period: During the run-in period (maximum 4 weeks prior tobaseline/randomization), subjects who meet the eligibility criteriaduring the screening visit will be enrolled in the study and will comeinto the clinic and undergo a hand grip strength test and 6-minute walktest (6 MWT) in order to familiarize the subjects with this test andthereby reduce test variability at the baseline visit. Subjects willalso be administered a breathlessness questionnaire before and after a 6MWT. In addition, during the run-in period subjects will undergobaseline echocardiography to assess cardiac function.

Blinded Treatment Phase: Part 1 (to be conducted in either the southernor northern hemisphere)

Treatment Phase (16 weeks): At the baseline visit (Visit 3), eligiblesubjects will be randomized to 1 of 3 treatment groups in a ratio of1:1:1

(1) BEZ235 5 mg daily

(2) BEZ235 10 mg daily

(3) Matching placebo daily

Subjects will be treated for 16 weeks with study drug. Approximately 56subjects will be enrolled in each treatment group for a total enrollmentof approximately 168 subjects. It is assumed that there will beapproximately a 10% drop-out rate, resulting in approximately 150subjects completing Part 1 of the study (approximately 50 subjects ineach arm). If the drop-out rate is higher than 10%, replacements may beenrolled.

At the baseline visit, subjects will undergo clinical evaluation andhave blood drawn for baseline safety assessments, EAST (IgE levels) andbiomarker assessments. Subjects who have not yet received their seasonalinfluenza vaccine will have influenza titers drawn. Subjects will alsoundergo grip strength, 6 MWT and complete a breathlessness questionnairebefore and after the 6 MWT at the baseline visit. Subjects will then begiven their first dose of study drug in the clinic and sent home withstudy drug. Subjects will also be sent home from the baseline visit witha diary. Subjects will be instructed to record the time and number ofcapsules of study medication administered at home each day in the diary.Subjects will also be instructed to record any infections symptoms thatoccur between visits in the diary. Finally, subjects will also be senthome with the short Wisconsin Upper Respiratory Symptom Survey(WURSS-21) that they will be instructed to fill out daily if theydevelop symptoms of an upper respiratory tract infection until theirsymptoms resolve.

After subjects have taken study drug for 1 week, a telephone callbetween the subjects and the site should take place to confirm that thefirst week of study drug was well tolerated. If study drug is nottolerated during the first week of study drug treatment, theinvestigator may discontinue study drug after discussion with theresTORbio Medical Monitor.

Provided the study drug continues to be well tolerated, subjects will betreated with study drug for 16 weeks, during which time they will returnto the clinic every 2 weeks (visits week 2, 4, 6 and 8) for the first 8weeks, and then every 4 weeks (visit week 12 and 16) for the final 8weeks to have clinical evaluations and blood drawn for safetyassessments. Blood will also be drawn for PK assessments at the week 4,6, and 8 visits. Blood will be drawn for biomarker assessments atbaseline and the week 6, 16 and 24 visits. Sites will contact subjectstwice weekly during the study either by phone or at the site visits toconfirm that the subjects are taking the study drug as instructed andthat the study drug continues to be well tolerated. Sites will alsoadminister a Respiratory Tract Infection Questionnaire during eachcontact and instruct subjects who develop 2 or more respiratory tractinfection symptoms or 1 or more non-respiratory tract infection symptomsto come in for an unscheduled visit as soon as possible so that they canbe evaluated by the Investigator.

Subjects who come to the site for an unscheduled visit due to infectionsymptoms will be evaluated by Investigators who will capture relevantsigns and symptoms of the infection on an Infection InvestigatorWorksheet as described in the Study Operations Manual. Subjects who havesymptoms of a RTI will have a nasopharyngeal swab taken in the clinic.If subjects are unable or unwilling to return to the site for anunscheduled visit, the Investigator can call the subject and completethe Infection Investigator Worksheet by phone and obtain thenasopharyngeal swab when the subject comes in for their next visit.Appropriate medical care will be provided for all infections. Subjectswho require additional medical care should be referred to theirhealthcare provider or to a hospital if indicated.

Subjects who receive a seasonal influenza vaccine as part of theirroutine health care during the study will be asked to return to the sitefor an unscheduled visit approximately 4 weeks after their influenzavaccination to have influenza titers drawn.

After 16 weeks of study drug treatment, subjects will have blood takenfor safety labs and biomarker assessments, and will undergo repeat gripstrength assessment, 6 MWT, and will complete a breathlessnessquestionnaire before and after the 6 MWT at the Week 16 visit. Subjectswill also have a repeat echocardiogram after completing 16 weeks ofstudy drug treatment.

Post Treatment Follow-up Period (8 weeks): All subjects will be followedup for 8 weeks after their last dose of study drug, during which timethey will be told to continue to record any infections that occur intheir diaries. Sites will continue to call subjects twice weekly andadminister the Twice Weekly Contact for Respiratory SymptomsQuestionnaire. Subjects who develop 2 or more respiratory tractinfection symptoms or 1 or more non-respiratory tract infection symptomswill be instructed to come in for an unscheduled visit as soon aspossible so that they can be evaluated by the Investigator as describedabove. Subjects with respiratory tract infection symptoms will have anasopharyngeal swab taken for respiratory virus PCR. Further details areprovided in the Study Operations Manual.

At the end of study Week 24 Visit, subjects will undergo a completephysical exam and have blood taken for safety labs and biomarkerassessments, and will undergo repeat grip strength assessment, 6 MWT,and will complete a breathlessness questionnaire before and after the 6MWT.

At or after the Week 24 visit, the investigator will review thesubjects' medical records to determine if additional infections occurredthat were not captured in the study database. Information aboutinfections that had not been captured will be recorded on theappropriate Investigator Infection Worksheet.

Patients who develop medically important laboratory abnormalities ormedically important AEs that are considered related to study drug andwhich are not resolved or stabilized at the end of the follow-up periodwill be followed, beyond the planned post treatment follow-up period,until these events have resolved or stabilized. If at the time of thecompletion of the study, these events are unresolved, they should becaptured as “ongoing” in the database.

Interim Analysis (IA): An IA will be performed when at least 120subjects randomized in Part 1 have completed the Week 16 visit. Anun-blinded Data Monitoring Committee (DMC) will review the results ofthe IA and make recommendations for the study. Possible decisions at theinterim analysis are:

whether to stop the study the BEZ235 monotherapy dosing arms forfutility, or continue with 1 or 2 BEZ235 treatment arms in Part 2;

whether to resize the study based on the rate of respiratory tractinfections during Part 1;

whether to change the duration of treatment based on safety data andrespiratory tract infection rate data from Part 1; and/or

whether additional PK sampling is needed and/or if the PK samplingregimen can be simplified.

Part 2 (to be conducted in the opposite hemisphere from Part 1): In Part2, between 310-430 subjects who meet the eligibility criteria during thescreening visit will be enrolled in the study. These subjects will berandomized to placebo, 0, 1 or 2 doses of BEZ235 from Part 1, and 5 mgor 10 mg BEZ235+RAD001 0.1 mg. Procedures identical to those in Part 1will apply during the screening, run-in, treatment and post treatmentfollow-up periods in Part 2.

FIG. 3-1 Study Design:

TABLE 1 Part 1 BEZ235 5 mg daily BEZ235 10 mg daily Placebo ↑Randomization ↑ Primary endpoint Screening Double-Blind TreatmentFollow-up 6 weeks 16 weeks 8 weeks  Run-in  4 weeks

TABLE 2 Part 2 BEZ235 dose(s) chosen from Part 1 BEZ235 + RAD001 0.1 mgdaily Placebo ↑ Randomization ↑ Primary endpoint Screening Double-BlindTreatment Follow-up 6 weeks 16 weeks 8 weeks  Run-in  4 weeks

Rationale for Study Design: The study is designed as a standardrandomized, blinded placebo-controlled, parallel-group study to obtainefficacy, tolerability and safety data in an unbiased fashion anddetermine the dose-response characteristics of the investigated drugs. Arun-in period is included to obtain baseline echocardiograms and tofamiliarize subjects with the 6 MWT and hand grip strength assessments.Sixteen weeks of treatment in Parts 1 and 2 are considered sufficient toresult in a clinically meaningful reduction in RTIs during winter coldand flu season in each hemisphere. The last 8 weeks of the study willprovide information about whether the reduction in RTIs persists aftersubjects have discontinued study drug treatment. PK samples will beobtained in all subjects at the week 4, 6, and 8 visits to enablecharacterization of PK after the study drugs have reached steady statelevels.

Study Population: Data from two previous clinical trials conducted byNovartis (CRAD002X2202 and CBEZ235Y2201) suggests that 6 weeks oftreatment of elderly subjects ≥65 years of age with low doses of BEZ235and/or RAD001 was safe and decreased the incidence of RTIs. Therisk/benefit of BEZ235 alone or in combination with RAD001 may be mostfavorable in elderly subjects who are at increased risk of RTI-relatedmorbidity and mortality. Therefore, in the current study we will enrollelderly subjects who are considered at increased risk of RTI-relatedmorbidity and mortality as defined by having one of the followingconditions:

-   -   Age ≥=85    -   Age >=65 years with:    -   Asthma    -   COPD Gold Class I or II        -   Postbronchodialator FEV₁/FVC<0.70 and FEV₁≥50% predicted    -   Chronic bronchitis    -   T2DM    -   CHF New York Heart Association functional classification I-II        -   No symptoms or mild symptoms (shortness of breath and/or            angina) and slight limitation during ordinary activity    -   Current smoker    -   One or more emergency room visits or hospitalizations for a RTI        during the previous 12 months

Further Inclusion Criteria:

Females must be post-menopausal. Women are considered post-menopausaland not of child bearing potential if they have had 12 months of natural(spontaneous) amenorrhea with an appropriate clinical profile (e.g. ageappropriate, history of vasomotor symptoms) or have had surgicalbilateral oophorectomy (with or without hysterectomy), totalhysterectomy or tubal ligation at least six weeks ago. In the case ofoophorectomy alone, only when the reproductive status of the woman hasbeen confirmed by follow up hormone level assessment is she considerednot of child bearing potential.

Sexually active male subjects with a partner of child-bearing potentialmust be willing to wear a condom while on study drug and for 1 weekafter stopping study drug, and should not father a child in this period.A condom is required to be used also by vasectomized men in order toprevent delivery of the drug via seminal fluid.

At screening and baseline, vital signs (systolic and diastolic bloodpressure, pulse rate and respiratory rate) will be assessed in a sittingposition after the subject has rested for at least three (3) minutes.Sitting vital signs should be within the following ranges:

Oral or tympanic body temperature between 35.0-37.5° C.

systolic blood pressure, 90-160 mm Hg

diastolic blood pressure, 50-95 mm Hg

pulse rate, 40-95 bpm

If vital signs are outside these ranges, the Investigator may obtain upto two additional readings, so that up to 3 consecutive assessments aremade. At least the last set of readings must be within the rangesprovided above in order for the subject to qualify.

Subjects must weigh at least 40 kg.

Subject must be able to communicate well with the investigator, and tounderstand and comply with the requirements of the study.

Results of a study may be quantified using different methods. Forinstance, results may be quantified by looking at the rate of infectionduring a particular time period as calculated by dividing the totalnumber of infections by the total number of subjects. Alternatively,results of a study may be quantified by looking at the percentage ofsubjects having one or more infections during a particular time period.

Primary Endpoint: The primary endpoint of the study is a decrease in thepercentage of subjects who experience one or more RTIs during the 16weeks of study drug treatment because RTIs cause significant morbidityand mortality in the elderly (Millett et al 2013).

Secondary Endpoints: As secondary endpoint, the percentage of subjectswho experience one or more RTIs for 24 weeks (16 weeks on study drugtreatment followed by 8 weeks off study drug treatment) will be assessedto determine if elderly subjects continue to have a reduced rate ofrespiratory tract infections after study drug discontinuation.

Since most respiratory tract infections in the elderly are caused byviruses (Jain et al 2015), the effect of BEZ235 alone or in combinationwith RAD001 on the incidence of laboratory-confirmed viral respiratorytract infections (as determined by respiratory virus PCR ofnasopharyngeal swabs) will be assessed during the 16 weeks subjects arereceiving study drug.

As an additional secondary endpoint, the pharmacokinetics (PK) of BEZ235given alone or in combination with RAD001 will be evaluated.

Exploratory Endpoints: As an exploratory endpoint, the effect of BEZ235alone or in combination with RAD001 on the incidence of RTIs will beexplored in specific subsets of the elderly to determine if the efficacyvaries between subgroups.

The effect of BEZ235 alone or in combination with RAD001 as compared toplacebo on the incidence of all infections as well as the incidence ofUTIs from baseline to Week 16 or 24 will also be explored because inclinical trial CBEZ235Y2201, elderly subjects treated for 6 weeks witheither BEZ235 10 mg daily or a combination of BEZ235 10 mg plus RAD0010.1 mg daily had a significant decrease in the rate of all infections aswell as a decrease in the rate UTIs over the year following study drugadministration.

As an additional exploratory endpoint, the effect of BEZ235 alone or incombination with RAD001 as compared to placebo on the duration and/orseverity of upper respiratory tract symptoms will be assessed using theWURSS-21 survey.

Results of CBEZ235Y2201 indicated that the response of elderlyvolunteers to influenza vaccination was enhanced when they received a 6week course of low dose BEZ235 in combination with RAD001 two weeksprior to receiving the influenza vaccine. Therefore as an exploratoryendpoint, the effect of BEZ235 alone or in combination with RAD001 ascompared to placebo on the response to influenza vaccination will beassessed in subjects who receive an influenza vaccine as part of theirroutine health care during the study.

mTOR inhibition by BEZ235 alone or in combination with RAD001 may haveanti-aging effects beyond just improving immune function and decreasinginfection rates. In our previous clinical trial, a subset of subjectstreated for 6 weeks with a combination of BEZ235 10 mg and RAD001 0.1 mgdaily, but not subjects treated with placebo, spontaneously reported anincrease in energy while on study drug. In addition, old mice treatedwith the mTOR inhibitor rapamycin have been noted to have increasedphysical activity and/or grip strength as compared to placebo-treatedcontrols (Flynn et al 2013). Therefore as an additional exploratoryendpoint, we will assess whether BEZ235 alone or in combination withRAD001 increases grip strength and/or endurance as assessed by a 6 MWT.

mTOR inhibitors have also been reported to improve cardiac function, inold mice (Flynn et al 2013). Therefore as additional exploratoryendpoints, we will determine if BEZ235 alone or in combination withRAD001 as compared to placebo improves cardiac function as assessed byechocardiography at Week 16 relative to baseline.

As a final exploratory endpoint, the effects of BEZ235 alone or incombination with RAD001 on gene expression in whole blood and on serumproteomics will be assessed to further elucidate the mechanisms by whichBEZ235 alone or in combination with RAD001 may improve immune functionand/or impact aging biology.

Rationale for Dose/Regimen, Route of Administration and Duration ofTreatment: In Novartis clinical study CBEZ235Y2201, all mTOR inhibitordosing regimens (BEZ235 10 mg daily, RAD001 0.5 mg or 0.1 mg daily, andBEZ235 10 mg daily plus RAD001 0.1 mg daily) were well tolerated andreduced the incidence of respiratory tract infections by between 50-60%during the 6 weeks elderly subjects received study drug treatment. Inaddition, the combination of BEZ235 10 mg and RAD001 0.1 mg dailysignificantly improved the response to influenza vaccination. In thecurrent study we will extend these findings and determine if BEZ235alone or in combination with RAD001 decreases the incidence ofrespiratory tract infections when elderly subjects are treated for alonger 16 week duration during winter cold and flu season in eachhemisphere when respiratory tract infection rates are the highest. Wewill also investigate the minimum efficacious dose level for BEZ235 bycomparing the safety and efficacy of BEZ235 5 mg vs 10 mg daily. BEZ23510 mg daily was chosen as a dose in the current study because it wasassociated with a reduction in both respiratory tract infections andtotal infections in clinical study CBEZ235Y2201. BEZ235 5 mg daily ispredicted by modeling and simulation to inhibit S6K phosphorylation byonly 10% and therefore may be sub-therapeutic and may establish theminimally efficacious dose in the current study. We will test doses ofBEZ235 above 10 mg up to BEZ235 10 mg twice daily, because PKvariability of BEZ235 in previously clinical studies was high at dosesof 25 mg and above. Accordingly, certain dosing regimens contemplatedherein comprise administration of, e.g., 10 mg BEZ235 twice a day. Insome such embodiments, a dosing regimen comprises administration of,e.g., 10 mg BEZ235 twice a day either alone (i.e., without RAD001) or incombination with RAD001, for instance in combination with a daily doseof 0.1 mg RAD001. In some embodiments, dosing regimens contemplatedherein comprise administering daily an amount of RAD001 ranging fromabout 0.015 mg to about 0.1 mg. In some such embodiments, an amount ofRAD001 ranging from about 0.015 mg to about 0.1 mg is administered incombination with an amount of BEZ235.

REFERENCES

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ENUMERATED EMBODIMENTS

In a first embodiment, the invention is a method comprising the step ofadministering (a) a low dose of RAD001 or a pharmaceutically acceptablesalt thereof; (b) a low dose of BEZ235 or a pharmaceutically acceptablesalt thereof; or (c) a combination thereof to a patient, wherein thepatient experiences fewer illnesses due to infection than a patient notadministered RAD001 or a pharmaceutically acceptable salt thereof,BEZ235 or a pharmaceutically acceptable salt thereof, or a combinationthereof.

In a second embodiment, the invention is a method according to the firstembodiment, wherein the patient's innate immunity is enhanced.

In a third embodiment, the invention is a method according to the firstor second embodiment, wherein antigen-specific immunity is not enhanced.

In a fourth embodiment, the invention is a method according to thesecond embodiment, wherein at least one interferon-inducing gene (ISG)is upregulated.

In a fifth embodiment, the invention is a method according to any one ofthe first through fourth embodiments, wherein the infection is a urinarytract infection; or an infection of the teeth or gums.

In a sixth embodiment, the invention is a method according to any one ofthe first through fourth embodiments, wherein the infection is arespiratory tract infection.

In a seventh embodiment, the invention is a method according to any oneof the first through fourth embodiments, wherein the infection is aviral infection.

In an eighth embodiment, the invention is a method according to any oneof the first through seventh embodiments, wherein the patient iselderly.

In a ninth embodiment, the invention is a method according to any one ofthe first through eighth embodiments, wherein the patient is at least 65years old.

In a tenth embodiment, the invention is a method according to any one ofthe first through ninth embodiments, wherein the patient is at least 75years old; or at least 85 years old.

In an eleventh embodiment, the invention is a method according to anyone of the first through tenth embodiments, wherein the low dose ofRAD001 or a pharmaceutically acceptable salt thereof, the low dose ofBEZ235 or a pharmaceutically acceptable salt thereof, or combinationthereof, is administered to the patient for up to about 6 consecutiveweeks.

In a twelfth embodiment, the invention is a method according to theeleventh embodiment, wherein the patient continues to experience fewerillnesses due to infection from about 1 day until about 1 year after theadministration of the RAD001 or a pharmaceutically acceptable saltthereof, the BEZ235 or a pharmaceutically acceptable salt thereof, orcombination thereof.

In a thirteenth embodiment, the invention is a method according to theeleventh embodiment, wherein the patient continues to experience fewerillnesses due to infection for at least 1 month and until about 1 yearafter the administration of the RAD001 or a pharmaceutically acceptablesalt thereof, the BEZ235 or a pharmaceutically acceptable salt thereof,or combination thereof.

In a fourteenth embodiment, the invention is a method according to theeleventh embodiment, wherein the patient continues to experience fewerillnesses due to infection for at least 3 months and until about 1 yearafter the administration of the RAD001 or a pharmaceutically acceptablesalt thereof, the BEZ235 or a pharmaceutically acceptable salt thereof,or combination thereof.

In a fifteenth embodiment, the invention is a method according to theeleventh embodiment, wherein the patient continues to experience fewerillnesses due to infection for at least 6 months after theadministration of the RAD001 or a pharmaceutically acceptable saltthereof, the BEZ235 or a pharmaceutically acceptable salt thereof, orcombination thereof.

In a sixteenth embodiment, the invention is a method according to anyone of the first through fifteenth embodiments, wherein the patient isadministered the low dose of BEZ235 or a pharmaceutically acceptablesalt thereof as a monotherapy.

In a seventeenth embodiment, the invention is a method according to anyone of the first through fifteenth embodiments, wherein the patient isadministered the low dose of RAD001 or a pharmaceutically acceptablesalt thereof as a monotherapy.

In an eighteenth embodiment, the invention is a method according to anyone of the first through fifteenth embodiments, wherein the patient isadministered both the low dose of RAD001 or a pharmaceuticallyacceptable salt thereof and the low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof as a combination therapy.

In a nineteenth embodiment, the invention is a method according to anyone of the first through sixteenth or eighteenth embodiments, whereinthe pharmacokinetic AUC variability of patients receiving the low doseof BEZ235 or a pharmaceutically acceptable salt thereof is lower than apatient receiving a higher dose of BEZ235 or a pharmaceuticallyacceptable salt thereof.

In a twentieth embodiment, the invention is a method according to anyone of the first through fifteenth, seventeenth, or eighteenthembodiments, wherein RAD001 or a pharmaceutically acceptable saltthereof is in the neutral form.

In a twenty-first embodiment, the invention is a method according to anyone of the first through sixteenth or eighteenth embodiments, whereinBEZ235 or a pharmaceutically acceptable salt thereof is the monotosylatesalt.

In a twenty-second embodiment, the invention is a method according toany one of the first through fifteenth or seventeenth throughtwenty-first embodiments, comprising the administration of 0.01-0.2 mgof RAD001 or a pharmaceutically acceptable salt thereof.

In a twenty-third embodiment, the invention is a method according to anyone of the first through sixteenth or eighteenth through twenty-secondembodiments, comprising the administration of 1-50 mg of BEZ235 or apharmaceutically acceptable salt thereof.

In a twenty-fourth embodiment, the invention is a method according toany one of the first through twenty-third embodiments, wherein thesubject is immunocompromised.

In a twenty-fifth embodiment, the invention is a method according to anyone of the first through twenty-fourth embodiments, wherein the subjecthas an impaired immune response.

In a twenty-sixth embodiment, the invention is a method according to anyone of the first through twenty-fifth embodiments, wherein the subjectis immunosenescent.

In a twenty-seventh embodiment, the invention is a method ofupregulating at least one interferon-inducing gene (ISG), comprisingadministering (a) a low dose of RAD001 or a pharmaceutically acceptablesalt thereof; (b) a low dose of BEZ235 or a pharmaceutically acceptablesalt thereof or (c) a combination thereof, to a patient.

In a twenty-eighth embodiment, the invention is a method of upregulatingat least one protein involved in extracellular matrix remodeling,comprising administering (a) a low dose of RAD001 or a pharmaceuticallyacceptable salt thereof; (b) a low dose of BEZ235 or a pharmaceuticallyacceptable salt thereof; or (c) a combination thereof, to a patient.

In a twenty-ninth embodiment, the invention is the method of thetwenty-eighth embodiment, wherein the protein involved in extracellularmatrix remodeling is significantly upregulated following treatment forat least about 1 week; at least about 2 weeks; at least about 3 weeks;or at least about 6 weeks.

In a thirtieth embodiment, the invention is a method of treating adisease or condition associated with aberrant extracellular matrixremodeling, comprising administering (a) a low dose of RAD001 or apharmaceutically acceptable salt thereof (b) a low dose of BEZ235 or apharmaceutically acceptable salt thereof; or (c) a combination thereof,to a patient.

In a thirty-first embodiment, the invention is the method of thethirtieth embodiment, wherein the disease or condition associated withaberrant extracellular matrix remodeling is selected from heart failure,heart failure with preserved ejection fraction, chronic renal failure,glomerunephropathy, skin aging, NASH, hepatitis fibrosis/cirrhosis,pulmonary fibrosis including idiopathic pulmonary fibrosis,aging-related tendon dysfunction/stiffening, arthritis includingosteoarthritis, sarcopenia, myelofibrosis, myelodysplasia, aging-relateddysfunction of the blood brain barrier, diabetic nephropathy,atherosclerosis, or wound healing.

We claim:
 1. A method comprising the step of administering daily as amonotherapy about 8-12 mg of BEZ235 or a pharmaceutically acceptablesalt thereof to a patient, wherein the patient experiences fewerillnesses due to respiratory tract infection than a patient notadministered BEZ235 or a pharmaceutically acceptable salt thereof. 2.The method according to claim 1, wherein the patient's innate immunityis enhanced.
 3. The method according to claim 2, whereinantigen-specific immunity is not enhanced.
 4. The method according toclaim 2, wherein at least one interferon-inducing gene (ISG) isupregulated.
 5. The method according to claim 1, wherein the respiratorytract infection is a viral respiratory tract infection.
 6. The methodaccording to claim 1, wherein the patient is elderly.
 7. The methodaccording to claim 1, wherein the patient is at least 65 years old. 8.The method according to claim 1, wherein the patient is at least 75years old.
 9. The method according to claim 1, wherein the patient is atleast 85 years old.
 10. The method according to claim 1, wherein theBEZ235 or a pharmaceutically acceptable salt thereof, is administered tothe patient for up to about 6 consecutive weeks.
 11. The methodaccording to claim 10, wherein the patient continues to experience fewerillnesses due to respiratory tract infection from about 1 day untilabout 1 year after the administration of the BEZ235 or apharmaceutically acceptable salt thereof.
 12. The method according toclaim 10, wherein the patient continues to experience fewer illnessesdue to respiratory tract infection for at least 1 month and until about1 year after the administration of the BEZ235 or a pharmaceuticallyacceptable salt thereof.
 13. The method according to claim 10, whereinthe patient continues to experience fewer illnesses due to respiratorytract infection for at least 3 months and until about 1 year after theadministration of the BEZ235 or a pharmaceutically acceptable saltthereof.
 14. The method according to claim 10, wherein the patientcontinues to experience fewer illnesses due to respiratory tractinfection for at least 6 months after the administration of the BEZ235or a pharmaceutically acceptable salt thereof.
 15. The method accordingto claim 1, wherein the pharmacokinetic AUC variability of patientsreceiving the BEZ235 or a pharmaceutically acceptable salt thereof islower than a patient receiving a higher dose of BEZ235 or apharmaceutically acceptable salt thereof.
 16. The method according toclaim 1, wherein BEZ235 or a pharmaceutically acceptable salt thereof isthe monotosylate salt.
 17. The method according to claim 1, wherein thesubject is immunocompromised.
 18. The method according to claim 1,wherein the subject has an impaired immune response.
 19. The methodaccording to claim 1, wherein the subject is immunosenescent.
 20. Themethod according to claim 1, wherein the BEZ235 or a pharmaceuticallyacceptable salt thereof is administered daily to the patient for up toabout 16 consecutive weeks.
 21. The method according to claim 1, whereinthe BEZ235 or a pharmaceutically acceptable salt thereof is administereddaily to the patient for up to about 24 consecutive weeks.
 22. Themethod according to claim 1, wherein about 9-11 mg of BEZ235 or apharmaceutically acceptable salt thereof is administered daily.
 23. Themethod according to claim 22, wherein about 10 mg of BEZ235 or apharmaceutically acceptable salt thereof is administered daily.